Aspartase variants, method of preparing the same and use thereof

Abstract

The present invention discloses an aspartase variant, method of preparing the same and use thereof. Compared with sequence 2 in the sequence listing, the amino acid sequence of the aspartase variant provided in the present invention has more than 96% identity, and there are mutations in T187I and N326C at positions 187 and 326, respectively, and has improved catalytic activity for the ammoniation of acrylic acid, compared with the aspartase shown in sequence 2. The experiment proves that the aspartase variant provided in the present invention does have improved catalytic activity for the ammoniation of acrylic acid compared with the wild type parent aspartase, and has better thermal stability and pH spectrum. The reaction can greatly increase the conversion ratio of acrylic acid.

Claims

1. An aspartase variant, the amino acid sequence thereof is more than 96% identical to SEQ ID NO. 2 in the sequence listing, and has mutations of T187I and N326C at both positions 187 and 326 of SEQ ID NO. 2, and the aspartase variant has improved catalytic activity for the ammoniation of acrylic acid compared with that of aspartase shown in SEQ ID NO. 2.

2. The aspartase variant according to claim 1, wherein compared with the SEQ ID NO. 2 in the sequence listing, the amino acid sequence of the aspartase variant further having a mutation at sequence position 156.

3. The aspartase variant according to claim 2, wherein compared with the SEQ ID NO. 2 in the sequence listing, the amino acid sequence of the aspartase variant further having a mutation at position L156F.

4. A nucleic acid molecule encoding the aspartase variant according to claim 1; or, a recombinant vector, expression cassette, or an isolated recombinant cell containing the nucleic acid molecule encoding the aspartase variant according to claim 1.

5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule is a gene encoding the aspartase variant, and compared with SEQ ID NO. 1 in the sequence listing, the nucleotide sequence of the gene has or just has the combined mutations shown in any one of 1) to 385) as follows: 1) T224A/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C; 2) T224A/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A11 43T; 3) T224A/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/C1367T/A1368G; 4) T224A/C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/A1216T/T1218G; 5) T224A/C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C; 6) T224A/C560T/G561T/G963T/A1216T/T1218G/A976T/A977G/T978C/C1276T/T1278C; 7) T224A/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T 1218G; 8) T224A/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A 1167C/A1216T/T1218G/C1276T/T1278C; 9) T224A/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 10) T224A/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 11) T224A/C560T/G561T/A853C/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 12) T224A/C560T/G561T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142 G/A1143T/C1276T/T1278C; 13) T224A/C560T/G561T/A677T/C678T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 14) T224A/C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C/C1366T/C1367T/A1368G; 15) T224A/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 16) T224A/C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C; 17) T224A/C560T/G561T/A611G/T612C/G963T/A976T/A977G/T978C; 18) T224A/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1216T/T1218G; 19) T224A/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1366T/C1367T/A1368G; 20) T224A/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G; 21) T224A/C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 22) T224A/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 23) T224A/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C; 24) T224A/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 25) T224A/C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 26) T224A/C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 27) T224A/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G; 28) T224A/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 29) T224A/C491T/A492T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/C1367T/A1368G; 30) T224A/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C; 31) T224A/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 32) T224A/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 33) T224A/C491T/A492T/C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/A1166T/A1167C; 34) T224A/C491T/A492T/C560T/G561T/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 35) T224A/C491T/A492T/C560T/G561T/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 36) T224A/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 37) T224A/A468T/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C; 38) T224A/A468T/C560T/G561T/A976T/A977G/T978C/C1276T/T1278C; 39) T224A/A468T/C560T/G561T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T 1278C; 40) T224A/A468T/C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 41) T224A/A468T/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1366T/C1367T/A1368G; 42) T224A/A468T/C560T/G561T/A853C/A976T/A977G/T978C/A1166T/A1167C; 43) T224A/A468T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C; 44) T224A/A468T/C560T/G561T/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 45) T224A/A468T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 46) T224A/A468T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 47) T224A/A468T/C560T/G561T/A611G/T612C/G963T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 48) T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C; 49) T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 50) T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 51) T224A/A468T/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C; 52) T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 53) T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 54) T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C; 55) T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G; 56) T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C; 57) T224A/A267T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 58) T224A/A267T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 59) T224A/A267T/C560T/G561T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G; 60) T224A/A267T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 61) T224A/A267T/C560T/G561T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T 1278C; 62) T224A/A267T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1166T/A1167C; 63) T224A/A267T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 64) T224A/A267T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 65) T224A/A267T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 66) T224A/A267T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 67) T224A/A267T/C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 68) T224A/A267T/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 69) T224A/A267T/C491T/A492T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 70) T224A/A267T/C491T/A492T/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 71) T224A/A267T/C491T/A492T/C560T/G561T/A976T/A977G/T978C; 72) T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/A977G/T978C; 73) T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G; 74) T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1166T/A1167C; 75) T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/C1276T/T1278C; 76) T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 77) T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 78) T224A/A267T/A468T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 79) T224A/A267T/A468T/C560T/G561T/G963T/A976T/A977G/T978C/C1276T/T1278C; 80) T224A/A267T/A468T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G; 81) T224A/A267T/A468T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C; 82) T224A/A267T/A468T/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 83) T224A/A267T/A468T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/C1367T/A1368G 84) T224A/A267T/A468T/C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 85) T224A/A267T/A468T/C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 86) T224A/A267T/A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C; 87) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 88) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/A1216T/T1218G; 89) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1216T/T1218G; 90) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 91) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C; 92) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C; 93) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 94) T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C; 95) C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1216T/T1218G; 96) C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T 1278C; 97) C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T 1218G/C1276T/T1278C; 98) C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C 1276T/T1278C; 99) C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A 1216T/T1218G/C1276T/T1278C; 100) C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T 1218G; 101) C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 102) C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 103) C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C; 104) C560T/G561T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 105) C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/C1276T/T1278C; 106) C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C; 107) C560T/G561T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 108) C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A 1143T; 109) C560T/G561T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 110) C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 111) C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 112) C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 113) C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 114) C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 115) C560T/G561T/A853C/G963T/A976T/A977G/T978C/C1276T/T1278C; 116) C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T 1278C; 117) C560T/G561T/A853C/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 118) C560T/G561T/A853C/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 119) C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216 T/T1218G/C1276T/T1278C; 120) C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166 T/A1167C/C1366T/C1367T/A1368G; 121) C560T/G561T/A677T/C678T/T772A/A774T/G963T/A976T/A977G/T978C/C1276T/T1278C; 122) C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/C1366T/C1367T/A1368G; 123) C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G/C1366T/C1367T/A1368G; 124) C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G; 125) C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 126) C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 127) C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A976T/A977G/T978C; 128) C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 129) C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 130) C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C; 131) C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/A1216T/T1218G/C 1276T/T1278C; 132) C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/A1166T/A1167C/A 1216T/T1218G; 133) C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C; 134) C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1216T/T1218G/C1366T/C1367T/A1368G; 135) C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1366T/C1367T/A1368G; 136) C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 137) C560T/G561T/A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A 1368G; 138) C560T/G561T/A677T/C678T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G; 139) C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/C1276T/T1278C; 140) C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/A1166T/A1167C/C 1276T/T1278C/C1366T/C1367T/A1368G; 141) C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/A1141C/A1142G/A 1143T/A1166T/A1167C; 142) C560T/G561T/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 143) C560T/G561T/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C; 144) C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/A977G/T978C/C1276T/T1278C; 145) C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 146) C560T/G561T/A611G/T612C/T772A/A774T/A971T/A972T/A976T/A977G/T978C; 147) C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 148) C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 149) C560T/G561T/A611G/T612C/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C; 150) C560T/G561T/A611G/T612C/G963T/A976T/A977G/T978C/A1141C/A1142G/A 1143T; 151) C560T/G561T/A611G/T612C/A976T/A977G/T978C/C1276T/T1278C/C1366T/C1367T/A1368G; 152) C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 153) C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1216T/T1218G/C1366T/C1367T/A1368G; 154) C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 155) C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 156) C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/C1366T/C1367T/A 1368G; 157) C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/A 1143T/C1276T/T1278C; 158) C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/A 1143T; 159) C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/G963T/A976T/A977G/T978C/A1216T/T1218G; 160) C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 161) C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A976T/A977G/T978C/C1276T/T1278C; 162) C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A976T/A977G/T978C; 163) C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 164) C560T/G561T/A611G/T612C/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 165) C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 166) C560T/G561T/A611G/T612C/A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G; 167) C560T/G561T/A611G/T612C/A677T/C678T/A853C/A976T/A977G/T978C/C1276T/T1278C; 168) C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G; 169) C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 170) C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G; 171) C491T/A492T/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 172) C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 173) C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 174) C491T/A492T/C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 175) C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/A1166T/A1167C; 176) C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A 1143T/C1276T/T1278C; 177) C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 178) C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 179) C491T/A492T/C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 180) C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/C1276T/T1278C; 181) C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 182) C491T/A492T/C560T/G561T/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1366T/C1367T/A1368G; 183) C491T/A492T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 184) C491T/A492T/C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/C1276T/T1278C; 185) C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 186) C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 187) C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/C1276T/T1278C; 188) C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G; 189) C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 190) C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 191) C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 192) C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 193) C491T/A492T/C560T/G561T/A468T/A971T/A972T/A976T/A977G/T978C; 194) A59T/C60G/T224A/C560T/G561T/A677T/C678T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 195) A59T/C60G/T224A/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/C1276T/T1278C; 196) A59T/C60G/T224A/C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C; 197) A59T/C60G/T224A/C560T/G561T/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 198) A59T/C60G/T224A/C491T/A492T/C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 199) A59T/C60G/T224A/C491T/A492T/C560T/G561T/A611G/T612C/A976T/A977G/T978C; 200) A59T/C60G/T224A/C491T/A492T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 201) A59T/C60G/T224A/A468T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C/C1366T/C1367T/A1368G; 202) A59T/C60G/T224A/A468T/C560T/G561T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 203) A59T/C60G/T224A/A468T/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 204) A59T/C60G/T224A/A468T/C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C/C1366T/C1367T/A1368G; 205) A59T/C60G/T224A/A468T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 206) A59T/C60G/T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 207) A59T/C60G/T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 208) A59T/C60G/T224A/A267T/C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C; 209) A59T/C60G/T224A/A267T/C560T/G561T/A611G/T612C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 210) A59T/C60G/T224A/A267T/C491T/A492T/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 211) A59T/C60G/T224A/A267T/C491T/A492T/C560T/G561T/A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 212) A59T/C60G/T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C; 213) A59T/C60G/T224A/A267T/A468T/C560T/G561T/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C; 214) A59T/C60G/T224A/A267T/A468T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 215) A59T/C60G/T224A/A267T/A468T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C; 216) A59T/C60G/T224A/A267T/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 217) A59T/C60G/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/C1276T/T1278C; 218) A59T/C60G/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1276T/T1278C; 219) A59T/C60G/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 220) A59T/C60G/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 221) A59T/C60G/C560T/G561T/T772A/A774T/A1216T/T1218G/A976T/A977G/T978C/C1276T/T1278C; 222) A59T/C60G/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G; 223) A59T/C60G/C560T/G561T/G963T/A976T/A977G/T978C/A1166T/A1167C; 224) A59T/C60G/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 225) A59T/C60G/C560T/G561T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 226) A59T/C60G/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 227) A59T/C60G/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 228) A59T/C60G/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 229) A59T/C60G/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 230) A59T/C60G/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 231) A59T/C60G/C560T/G561T/A853C/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 232) A59T/C60G/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A11 43T; 233) A59T/C60G/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 234) A59T/C60G/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G; 235) A59T/C60G/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 236) A59T/C60G/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1166T/A 1167C; 237) A59T/C60G/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A 1142G/A1143T; 238) A59T/C60G/C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/C1276T/T1278C; 239) A59T/C60G/C560T/G561T/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 240) A59T/C60G/C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 241) A59T/C60G/C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 242) A59T/C60G/C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C; 243) A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 244) A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 245) A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 246) A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 247) A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 248) A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1166T/A1167C; 249) A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 250) A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C; 251) A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 252) A59T/C60G/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1166T/A1167C/C1366T/C1367T/A1368G; 253) A59T/C60G/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 254) A59T/C60G/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C; 255) A59T/C60G/C491T/A492T/C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 256) A59T/C60G/C491T/A492T/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 257) A59T/C60G/C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C; 258) A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/C1276T/T1278C; 259) A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C; 260) A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 261) A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C; 262) A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/A971T/A972T/A976T/A977G/T978C; 263) A59T/C60G/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 264) A59T/C60G/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 265) A59T/C60G/A468T/C560T/G561T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C; 266) A59T/C60G/A468T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 267) A59T/C60G/A468T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/C1367T/A1368G; 268) A59T/C60G/A468T/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 269) A59T/C60G/A468T/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 270) A59T/C60G/A468T/C560T/G561T/A853C/A976T/A977G/T978C; 271) A59T/C60G/A468T/C560T/G561T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 272) A59T/C60G/A468T/C560T/G561T/A677T/C678T/A976T/A977G/T978C; 273) A59T/C60G/A468T/C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T; 274) A59T/C60G/A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 275) A59T/C60G/A468T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C; 276) A59T/C60G/A468T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 277) A59T/C60G/A468T/C491T/A492T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T 1278C; 278) A59T/C60G/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 279) A59T/C60G/A468T/C491T/A492T/C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C; 280) A59T/C60G/A468T/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 281) A59T/C60G/A468T/C491T/A492T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 282) A59T/C60G/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 283) A59T/C60G/A267T/C560T/G561T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 284) A59T/C60G/A267T/C560T/G561T/T772A/A774T/A976T/A977G/T978C; 285) A59T/C60G/A267T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C; 286) A59T/C60G/A267T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 287) A59T/C60G/A267T/C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/C1276T/T1278C; 288) A59T/C60G/A267T/C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T; 289) A59T/C60G/A267T/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1366T/C1367T/A1368G; 290) A59T/C60G/A267T/C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 291) A59T/C60G/A267T/C491T/A492T/C560T/G561T/A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1366T/C1367T/A1368G; 292) A59T/C60G/A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C; 293) A59T/C60G/A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 294) A59T/C60G/A267T/A468T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1366T/C1367T/A1368G; 295) A59T/C60G/A267T/A468T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 296) A59T/C60G/A267T/A468T/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 297) A59T/C60G/A267T/A468T/C560T/G561T/A853C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 298) A59T/C60G/A267T/A468T/C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 299) A59T/C60G/A267T/A468T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 300) A59T/C60G/A267T/A468T/C560T/G561T/A611G/T612C/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 301) A59T/C60G/A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 302) A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 303) A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 304) A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 305) A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 306) A468T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C; 307) A468T/C560T/G561T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 308) A468T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G; 309) A468T/C560T/G561T/G963T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 310) A468T/C560T/G561T/A853C/A976T/A977G/T978C/C1276T/T1278C; 311) A468T/C560T/G561T/A853C/A976T/A977G/T978C/A1166T/A1167C; 312) A468T/C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G; 313) A468T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G; 314) A468T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/A1142G/A 1143T; 315) A468T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 316) A468T/C560T/G561T/A611G/T612C/G963T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 317) A468T/C560T/G561T/A611G/T612C/G963T/A971T/A972T/A976T/A977G/T978C, 318) A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/C1276T/T1278C; 319) A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1141C/A1142G/A 1143T/C1276T/T1278C; 320) A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1141C/A1142G/A 1143T; 321) A468T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 322) A468T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 323) A468T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/G963T/A976T/A97 7G/T978C/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 324) A468T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/C1367T/A1368G; 325) A468T/C560T/G561T/A611G/T612C/A1166T/A1167C/A976T/A977G/T978C/C 1276T/T1278C; 326) A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C; 327) A468T/C491T/A492T/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 328) A468T/C491T/A492T/C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/T978C; 329) A468T/C491T/A492T/C560T/G561T/A976T/A977G/T978C/A1166T/A1167C; 330) A468T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/C1276T/T1278C; 331) A468T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 332) A468T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 333) A468T/C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/C1276T/T1278C; 334) A468T/C491T/A492T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 335) A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C; 336) A267T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/C1276T/T1278C; 337) A267T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/C1366T/C1367T/A 1368G; 338) A267T/C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 339) A267T/C560T/G561T/T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G; 340) A267T/C560T/G561T/A976T/A977G/T978C/C1276T/T1278C/C1366T/C1367T/A1368G; 341) A267T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 342) A267T/C560T/G561T/A853C/A976T/A977G/T978C/C1276T/T1278C; 343) A267T/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T; 344) A267T/C560T/G561T/A677T/C678T/T772A/A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G; 345) A267T/C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C; 346) A267T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 347) A267T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/A972T/A976T/A977G/T978C; 348) A267T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 349) A267T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/C1366T/C1367T/A 1368G; 350) A267T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1216T/T1218G; 351) A267T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 352) A267T/C560T/G561T/A611G/T612C/A677T/C678T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 353) A267T/C560T/G561T/A611G/T612C/A677T/C678T/A976T/A977G/T978C/A1166T/A1167C; 354) A267T/C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/T1218G; 355) A267T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C; 356) A267T/C491T/A492T/C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C; 357) A267T/C491T/A492T/C560T/G561T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G; 358) A267T/C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C; 359) A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 360) A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 361) A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/T978C; 362) A267T/C491T/A492T/C560T/G561T/A611G/T612C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C; 363) A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/G963T/A976T/A977G/T978C/C1276T/T1278C; 364) A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C; 365) A267T/A468T/C560T/G561T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C; 366) A267T/A468T/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 367) A267T/A468T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 368) A267T/A468T/C560T/G561T/A677T/C678T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 369) A267T/A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/T1278C/C1366T/C1367T/A1368G; 370) A267T/A468T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/C1276T/T1278C/C1366T/C1367T/A1368G; 371) A267T/A468T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 372) A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 373) A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/T978C; 374) A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G; 375) A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 376) A267T/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 377) A267T/A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1166T/A1167C; 378) A267T/A468T/C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C; 379) A267T/A468T/C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C; 380) A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G; 381) A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T; 382) A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C; 383) A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/A1166T/A1167C; 384) A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C/C1366T/C1367T/A1368G; 385) A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T.

6. A method of producing a target product, comprising the steps of using acrylic acid and an ammonia-containing material to react under the catalysis of the aspartase variant according to claim 1 to obtain the target product; and the target product is selected from any one or more of the following: β-alanine, β-alanine salt, multimer of β-alanine.

7. The method according to claim 6, wherein the ammonia-containing material is ammonia water.

8. The method according to claim 6, wherein in the reaction system, the concentration of acrylic acid or acrylate is 20-400 mM, the pH value is 7.0-10.5, and the reaction temperature is 30-55° C., the reaction time is 0.5-5h.

9. The aspartase variant according to claim 3, wherein compared with the SEQ ID NO. 2 in the sequence listing, the amino acid sequence of the aspartase variant has or just has the combined mutations shown in any one of 1)-127) as follows: 1) D20V/Q89H/L156F/T164I/T187I/Y204C/L258I/M285L/N326C/K381R/R426C; 2) Q89H/L156F/T164I/T187I/L258I/N326C/K389I/I406L/R426C; 3) L156F/T164I/T187I/L258I/K324I/N326C/K389I; 4) D20V/L156F/T164I/T187I/Y204C/N226I/M321I/K324I/N326C/K389I/R426C; 5) L156F/T187I/Y204C/K324I/N326C/K389I/I406L; 6) D20V/Q89H/L156F/T187I/M285L/N326C/K381R/K389I; 7) L156F/T164I/T187I/L258I/N326C/I406L/R426C; 8) L156F/T187I/N226I/L258I/N326C/I406L; 9) D20V/L156F/T164I/T187I/N226I/M285L/M321I/K324I/N326C/I406L/R426C; 10) V75E/Q89H/L156F/T187I/N326C/K381R/R426C; 11) V75E/L156F/T187I/M285L/M321I/K324I/N326C/K381R/P456L; 12) Q89H/L156F/T164I/T187I/Y204C/N226I/M285L/K324I/N326C/K381R; 13) V75E/L156F/T187I/N226I/L258I/M285L/N326C; 14) V75E/Q89H/L156F/T164I/T187I/N226I/N326C/K389I/I406L; 15) D20V/Q89H/L156F/T187I/Y204C/M285L/K324I/N326C/K381R/K389I; 16) D20V/Q89H/L156F/T187I/N226I/N326C/K381R/K389I; 17) D20V/V75E/L156F/T187I/N226I/M321I/K324I/N326C/R426C/P456L; 18) V75E/L156F/T187I/Y204C/M321I/N326C/K389I/R426C; 19) D20V/L156F/T187I/L258I/M321I/K324I/N326C; 20) V75E/Q89H/L156F/T187I/M321I/N326C/R426C; 21) V75E/L156F/T164I/T187I/Y204C/N226I/L258I/N326C/I406L; 22) V75E/Q89H/L156F/T164I/T187I/Y204C/M285L/N326C/K381R/K389I/R426C; 23) D20V/V75E/L156F/T187I/N326C/K381R/R426C; 24) D20V/Q89H/L156F/T187I/N226I/M321I/N326C/K381R/I406L; 25) V75E/L156F/T164I/T187I/L258I/M285L/M321I/N326C/K381R/K389I/R426C; 26) D20V/V75E/Q89H/L156F/T187I/L258I/M285L/K324I/N326C; 27) L156F/T164I/T187I/K324I/N326C/R426C; 28) D20V/V75E/L156F/T164I/T187I/L258I/M321I/K324I/N326C/K381R/I406L; 29) V75E/Q89H/L156F/T187I/M321I/K324I/N326C; 30) L156F/T164I/T187I/K324I/N326C/K381R; 31) L156F/T164I/T187I/L258I/K324I/N326C; 32) D20V/V75E/Q89H/L156F/T187I/Y204C/L258I/M321I/N326C/K389I/I406L; 33) D20V/L156F/T187I/M285L/M321I/K324I/N326C/K389I; 34) D20V/L156F/T187I/Y204C/N326C/K389I/I406L/R426C; 35) D20V/V75E/L156F/T187I/N326C/K389I/I406L/R426C; 36) D20V/Q89H/L156F/T164I/T187I/L258I/M285L/N326C/K381R/R426C; 37) V75E/Q89H/L156F/T187I/M321I/N326C/I406L; 38) D20V/Q89H/L156F/T164I/T187I/N226I/M321I/N326C/K389I/I406L/R426C; 39) L156F/T187I/Y204C/M321I/K324I/N326C; 40) D20V/L156F/T164I/T187I/M321I/K324I/N326C; 41) L156F/T187I/Y204C/N226I/M285L/M321I/N326C/I406L/R426C/P456L; 42) V75E/Q89H/L156F/T187I/K324I/N326C/K389I/R426C/P456L; 43) L156F/T164I/T187I/K324I/N326C/K381R/I406L/R426C; 44) D20V/Q89H/L156F/T164I/T187I/Y204C/M285L/K324I/N326C/K381R/R426C; 45) L156F/T187I/Y204C/N326C/R426C; 46) V75E/Q89H/L156F/T187I/L258I/M321I/N326C/I406L/R426C; 47) L156F/T187I/N226I/N326C/K381R; 48) L156F/T187I/L258I/M285L/M321I/K324I/N326C/K381R/I406L/R426C; 49) D20V/L156F/T187I/N226I/M285L/N326C/K381R; 50) D20V/Q89H/L156F/T187I/M285L/K324I/N326C/K389I; 51) Q89H/L156F/T164I/T187I/Y204C/N226I/L258I/M285L/N326C/K389I/R426C; 52) L156F/T164I/T187I/N326C/K389I; 53) L156F/T164I/T187I/N226I/N326C/R426C; 54) V75E/L156F/T164I/T187I/L258I/M285L/N326C/K381R/K389I/R426C; 55) V75E/L156F/T164I/T187I/Y204C/L258I/N326C/K389I/I406L; 56) D20V/Q89H/L156F/T187I/K324I/N326C/K381R/R426C; 57) Q89H/L156F/T187I/Y204C/N226I/M285L/M321I/N326C/K389I/R426C; 58) Q89H/L156F/T164I/T187I/L258I/N326C/K389I; 59) Q89H/L156F/T164I/T187I/Y204C/N226I/K324I/N326C/P456L; 60) V75E/L156F/T164I/T187I/Y204C/L258I/M285L/M321I/N326C/K389I/I406L; 61) Q89H/L156F/T164I/T187I/N226I/L258I/M321I/K324I/N326C; 62) D20V/L156F/T187I/M285L/M321I/K324I/N326C/R426C; 63) V75E/Q89H/L156F/T164I/T187I/Y204C/N226I/M285L/K324I/N326C; 64) V75E/L156F/T164I/T187I/Y204C/M285L/N326C; 65) V75E/Q89H/L156F/T187I/Y204C/N326C; 66) L156F/T187I/M321I/N326C/I406L; 67) V75E/Q89H/L156F/T164I/T187I/Y204C/L258I/M285L/M321I/N326C/K389I; 68) Q89H/L156F/T164I/T187I/Y204C/N226I/L258I/M285L/N326C/K389I; 69) Q89H/L156F/T187I/Y204C/N226I/M321I/K324I/N326C/K381R/I406L; 70) L156F/T187I/M285L/N326C/R426C; 71) V75E/L156F/T187I/Y204C/L258I/M321I/K324I/N326C/K381R/I406L/R426C; 72) D20V/L156F/T187I/M285L/K324I/N326C/K381R/K389I; 73) Q89H/L156F/T164I/T187I/Y204C/M285L/K324I/N326C/K381R; 74) V75E/L156F/T187I/N326C/K389I/R426C; 75) L156F/T187I/Y204C/K324I/N326C/K381R/R426C; 76) T164I/T187I/L156F/K324I/N326C; 77) Q89H/L156F/T187I/Y204C/N326C/K381R/I406L/R426C/P456L; 78) L156F/T187I/L258I/M321I/N326C; 79) D20V/L156F/T187I/N226I/N326C; 80) V75E/Q89H/L156F/T187I/N226I/M321I/K324I/N326C/R426C; 81) D20V/L156F/T187I/M285L/N326C; 82) V75E/Q89H/L156F/T164I/T187I/Y204C/M285L/M321I/N326C/K389I; 83) L156F/T187I/Y204C/N226I/M285L/K324I/N326C/K389I/R426C/P456L; 84) D20V/L156F/T187I/Y204C/N226I/L258I/K324I/N326C/K381R/R426C; 85) D20V/L156F/T187I/Y204C/K324I/N326C; 86) L156F/T187I/Y204C/L258I/M285L/M321I/N326C/K381R/K389I; 87) V75E/Q89H/L156F/T164I/T187I/M285L/N326C/K381R/K389I/R426C; 88) D20V/L156F/T187I/M321I/N326C/I406L/R426C; 89) V75E/Q89H/L156F/T187I/N226I/M285L/N326C/K381R/K389I; 90) D20V/L156F/T164I/T187I/L258I/N326C/K381R/K389I/I406L/R426C; 91) D20V/V75E/Q89H/L156F/T164I/T187I/L258I/N326C/K381R/K389I/I406L; 92) L156F/T187I/M321I/N326C/K389I/R426C; 93) Q89H/L156F/T187I/N226I/L258I/M285L/N326C/K389I/I406L/R426C; 94) Q89H/L156F/T187I/Y204C/M285L/M321I/N326C/R426C/P456L; 95) Q89H/L156F/T187I/N226I/K324I/N326C/K389I/I406L/R426C; 96) L156F/T164I/T187I/Y204C/N326C/K381R/R426C; 97) Q89H/L156F/T187I/Y204C/N226I/L258I/M285L/M321I/K324I/N326C; 98) V75E/L156F/T164I/T187I/L258I/N326C; 99) L156F/T187I/Y204C/N326C/K381R/R426C; 100) V75E/L156F/T187I/Y204C/L258I/M321I/N326C/K381R/I406L/R426C; 101) D20V/L156F/T164I/T187I/L258I/M321I/N326C/K381R/K389I/I406L/R426C; 102) D20V/L156F/T187I/M321I/K324I/N326C/K389I/R426C/P456L; 103) V75E/L156F/T187I/N226I/K324I/N326C/I406L/R426C; 104) Q89H/L156F/T187I/Y204C/M285L/M321I/N326C/K389I/I406L; 105) D20V/Q89H/L156F/T187I/L258I/M321I/N326C/K381R/P456L; 106) L156F/T187I/Y204C/M321I/N326C/K389I/R426C; 107) D20V/V75E/L156F/T187I/Y204C/M285L/N326C/K381R/K389I; 108) D20V/V75E/L156F/T187I/M321I/K324I/N326C/R426C/P456L; 109) V75E/Q89H/L156F/T164I/T187I/N226I/M321I/N326C/I406L; 110) L156F/T187I/Y204C/K389I/N326C/R426C; 111) Q89H/L156F/T164I/T187I/Y204C/M285L/M321I/N326C/K389I/I406L; 112) Q89H/L156F/T164I/T187I/M285L/N326C/K389I/I406L/R426C; 113) V75E/L156F/T164I/T187I/M321I/N326C; 114) D20V/Q89H/L156F/T187I/Y204C/N226I/M285L/K324I/N326C/K381R/K389I; 115) D20V/V75E/Q89H/L156F/T187I/Y204C/M285L/M321I/N326C/K389I; 116) L156F/T164I/T187I/Y204C/N226I/L258I/M321I/K324I/N326C; 117) L156F/T187I/N226I/L258I/M285L/M321I/N326C/K381R/K389I/I406L; 118) V75E/L156F/T187I/M285L/N326C/K389I; 119) Q89H/L156F/T187I/Y204C/N226I/L258I/N326C/K389I/I406L/R426C; 120) D20V/V75E/L156F/T164I/T187I/Y204C/M285L/N326C/K381R/K389I; 121) V75E/Q89H/L156F/T164I/T187I/N226I/N326C/I406L; 122) Q89H/L156F/T187I/L258I/M321I/K324I/N326C; 123) D20V/L156F/T164I/T187I/N226I/L258I/M285L/N326C/I406L/R426C; 124) L156F/T187I/Y204C/N326C/K381R; 125) V75E/L156F/T164I/T187I/Y204C/N226I/L258I/M285L/M321I/K324I/N326C; 126) Q89H/L156F/T187I/N326C/K381R; or 127) V75E/L156F/T187I/N326C/R426C.

Description

BEST MODE OF IMPLEMENTING THE INVENTION

(1) The following examples are provided for better understanding of the present invention, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples all carried out three times, and the results were averaged.

Example 1. Preparation and Identification of Aspartase Variants

(2) This example involves a total of 300 aspartase variants, all of which are derived from a wild type aspartase protein mature body (corresponding encoding gene is shown in sequence 1 in sequence listing) by mutations. The mutation sites of the 300 aspartase variants at the protein and gene levels are specifically shown in Table 1.

(3) I. Cloning

(4) A method known in the art is used to construct a plasmid containing the gene encoding the aspartase and its variant, and the resulting recombinant plasmid is transformed into a suitable host cell.

(5) In this example, the vector plasmid used is specifically pET21a. The gene fragment containing gene encoding the aspartase and variant thereof are used as templates, TATGGCTAGCATGACTGGTatgaataccgatgttcgtattg and GCTAGTTATTGCTCAGCGGttttctctccagcaattcccg as primers to amplify the corresponding nucleotide gene fragments by PCR. Then, the gene fragment obtained by PCR amplification and pET21a are mixed at a molar ratio of 1:1, and the method of Gibson assembly is used (see, Ji Zhicheng et al., Using the Gibson Assembly method to construct a plant expression vector, Journal of south China agricultural university, 2014, 35(5): 112-116) to construct a plasmid containing a gene encoding aspartase or a variant thereof. E. coli BL21 (DE3) is used as a host cell, and a recombinant plasmid containing the gene encoding aspartase was introduced into the host cell.

(6) II. Expression

(7) Host cells containing the recombinant plasmid constructed in step I were cultured using a self-inducing medium. The composition of the self-induction medium is as follows:10 g/L peptone, 5 g/L yeast powder, 3.55 g/L disodium hydrogen phosphate, 3.4 g/L potassium dihydrogen phosphate, 2.68 g/L ammonium chloride, 0.71 g/L sodium sulfate, 0.493 g/L magnesium sulfate heptahydrate, 0.27 g/L ferric chloride hexahydrate, 20 mL/L 100% glycerol, 0.5 g/L glucose, 2 g/L lactose, 50 mg/L ampicillin sodium. Host cells containing the recombinant plasmid are inoculated into a self-inducing medium, and fermentation is performed in a batch fermentation manner. Incubate with shaking at 30° C. and 200 rpm for 20 hours.

(8) III. Cell Collection

(9) The following three methods may be used: {circle around (1)} Centrifugation: centrifuging the cell culture solution at 4000 g for 10 minutes to collect cells; {circle around (2)} Hollow fiber membrane filtration: filtering the cell culture solution with a 0.22 micron hollow fiber membrane to collect cells; {circle around (3)} Ceramic membrane filtration: collecting cells by filtering through a 50 KDa ceramic membrane. Under laboratory conditions, cells are preferably collected by centrifugation (this method is used in this example), and the enzyme activity is compared.

(10) IV. Purification of Expressed Proteins

(11) Resuspend the cells with ultrapure water to an OD.sub.550 value of 200. Cells were disrupted by ultrasonication. The conditions of ultrasonication were as follows: working for 1 second, intermittent for two seconds, 180 W, total disruption for 20-25 minutes. Cells may also be lysed using a high pressure homogeneous fragmentation method. The conditions for high pressure homogeneous fragmentation were as follows: 50 HZ, 800 bar, and fragmentation for 2 times.

(12) First, high-speed centrifugation was used to remove cell debris and macromolecular impurities. The centrifugation conditions were as follows: taking 10-50 mL of cell disruption solution, centrifuging for 20-40 minutes at 12000 rpm, 4° C. Then, ultracentrifugation was used to concentrate the supernatant from which cell debris and macromolecular impurities have been removed. The selected ultrafiltration centrifuge tube was Amicon Ultra-15 (or 50) mL ultracentrifugal ultracentrifuge equipped with Ultracel-50 ultrafiltration membrane filter tube. The centrifugation conditions were as follows: centrifuging for 20-40 minutes at 12000 rpm, 4° C. 1-5 mL of pure water was added at 4° C. into the ultrafiltration tube, so as to resuspend the aspartase protein retained by the ultrafiltration tube.

(13) V. Determination of Enzyme Protein Concentration

(14) After the enzyme protein suspension was diluted 100-500 times, its concentration was measured by Bradford method.

(15) VI. Determination of Catalytic Activity for the Ammoniation of Acrylic Acid

(16) Preparation of substrates affected by aspartase variants: acrylic acid or acrylate 20-400 mM, adjust the pH to 7.0-9.5 with ammonia.

(17) The operation steps for the determination of aspartase variants enzyme activity are as follows: take a certain amount of fermented cells or purified enzyme protein, add a substrate, and react at a certain temperature for 0.5-5 h.

(18) β-alanine was detected by high performance liquid chromatography, and the amount of β-alanine before and after the reaction was determined by external standard method. The conditions for liquid chromatography detection are as follows: chromatographic column of Eclipse XDB-C18; mobile phase, potassium dihydrogen phosphate (pH 2.6): acetonitrile=95:5 (volume ratio); flow rate 1.0 mL/min; and detection wavelength of 210 nm.

(19) Preparation of potassium dihydrogen phosphate (KH.sub.2PO.sub.4) with pH of 2.6: take 1.44 g of KH.sub.2PO.sub.4 and dissolve it in 1 L of ultrapure water, and adjust the pH to 2.6 with 0.6-0.7 mL of 85% phosphoric acid.

(20) Definition of the activity of enzymes for catalyzing the ammoniation of acrylic acid: the production of 1 mM of β-alanine within 1 minute is defined as 1 U. Definition of the activity of enzymes for catalyzing the ammoniation of acrylic acid by unit cell: the production of 1 mM of β-alanine within 1 minute at the concentration of 1.0 OD.sub.550 cell is defined as 1 U/OD.sub.550. Definition of the activity of enzymes for catalyzing the ammoniation of acrylic acid by unit protein: the production of 1 mM of β-alanine within 1 minute using 1.0 mg of aspartase protein is defined as 1 U/mg.

(21) Calculation of Conversion Ratio:

(22) Conversion ratio %=A/B×100%, where A is the molar concentration of produced β-alanine in mM, and B is the initial molar concentration of acrylic acid in the unit of mol/L.

(23) Calculation of Enzyme Activity Per Unit Cell:

(24) Enzyme activity per unit cell=molar concentration of produced β-alanine/reaction time/concentration of cell, where the unit of reaction time is min, and the cell concentration is OD.sub.550 value in the reaction system.

(25) Calculation of Enzyme Activity Per Unit Protein:

(26) Enzyme activity per unit protein=molar concentration of produced β-alanine/reaction time/concentration of protein, where the unit of reaction time is min, and the concentration of protein is the crude enzyme protein concentration in the reaction system, in the unit of g/L.

(27) VII. Relative Enzyme Activity

(28) Mixing 25 mg of the crude enzyme protein of wild type aspartase or a variant thereof with 1.0 mL of a 400 mM substrate at pH 8.0, stirring at 40° C. for 1.0 h of reaction, and then its catalytic activity of ammoniation of acrylic acid is measured.

(29) Relative enzyme activity is the ratio of the activity of the aspartase variant enzyme activity to the wild type aspartase enzyme activity. The results of the relative enzyme activity of the aspartase variants are shown in Table 1.

(30) TABLE-US-00002 TABLE 1 Aspartase variant protein, gene mutation sites and the relative results of the catalytic activity of the ammoniation of acrylic acid relative activity Variants/ Variants substitutions WT WT — — WT AHB0 protein: T187I/N326C 5.42 01 gene: C560T/G561T/A976T/A977G/T978C AHB0 protein: V75E/Q89H/T187I/L258I/N326C/K381R/K389I 12.77 02 gene: T224A/A267T/C560T/G561T/T772A/A774T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/A1166T/A1167C AHB0 protein: D20V/Q89H/L156F/T164I/T187I/Y204C/L258I/M285L/ 28.59 03 N326C/K381R/R426C gene: A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/ A611G/T612C/T772A/A774T/A853C/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB0 protein: Q89H/L156F/T164I/T187I/L258I/N326C/K389I/I406L/R426C 28.05 04 gene: A267T/A468T/C491T/A492T/C560T/G561T/T772A/A774T/ A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/ C1276T/T1278C AHB0 protein: L156F/T187I/Y204C/K324I/N326C/K389I/I406L 26.98 05 gene: A468T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/ A977G/T978C/A1166T/A1167C/A1216T/T1218G AHB0 protein: T187I/N226I/M285L/M321I/K324I/N326C/K381R/K389I/ 13.95 06 I406L/P456L gene: C560T/G561T/A677T/C678T/A853C/G963T/A971T/A972T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C/A1216T/T1218G/C1366T/C1367T/A1368G AHB0 protein: D20V/Q89H/L156F/T187I/M285L/N326C/K381R/K389I 21.02 07 gene: A59T/C60G/A267T/A468T/C560T/G561T/A853C/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C AHB0 protein: D20V/Q89H/T164I/T187I/Y204C/N226I/K324I/N326C 39.97 08 gene: A59T/C60G/A267T/C491T/A492T/C560T/G561T/A611G/ T612C/A677T/C678T/A971T/A972T/A976T/A977G/T978C AHB0 protein: L156F/T164I/T187I/L258I/N326C/I406L/R426C 31.14 09 gene: A468T/C491T/A492T/C560T/G561T/T772A/A774T/A976T/ A977G/T978C/A1216T/T1218G/C1276T/T1278C AHB0 protein: V75E/T187I/N226I/L258I/K324I/N326C/K381R/I406L 36.16 10 gene: T224A/C560T/G561T/A677T/C678T/T772A/A774T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1216T/T1218G AHB0 protein: V75E/T187I/L258I/M285L/N326C/I406L 6.70 11 gene: T224A/C560T/G561T/T772A/A774T/A853C/A976T/A977G/ T978C/A1216T/T1218G AHB0 protein: L156F/T187I/N226I/L258I/N326C/I406L 22.48 12 gene: A468T/C560T/G561T/A677T/C678T/T772A/A774T/A976T/ A977G/T978C/A1216T/T1218G AHB0 protein: D20V/L156F/T164I/T187I/N226I/M285L/M321I/K324I/ 6.87 13 N326C/I406L/R426C gene: A59T/C60G/A468T/C491T/A492T/C560T/G561T/A677T/ C678T/A853C/G963T/A971T/A972T/A976T/A977G/T978C/ A1216T/T1218G/C1276T/T1278C AHB0 protein: V75E/T187I/N226I/M321I/K324I/N326C/P456L 11.06 14 gene: T224A/C560T/G561T/A677T/C678T/G963T/A971T/A972T/ A976T/A977G/T978C/C1366T/C1367T/A1368G AHB0 protein: V75E/Q89H/L156F/T187I/N326C/K381R/R426C 22.81 15 gene: T224A/A267T/A468T/C560T/G561T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB0 protein: T187I/N226I/M321I/N326C/K389I/I406L 14.50 16 gene: C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/ A1166T/A1167C/A1216T/T1218G AHB0 protein: V75E/L156F/T187I/M285L/M321I/K324I/N326C/K381R/P456L 20.35 17 gene: T224A/A468T/C560T/G561T/A853C/G963T/A971T/A972T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/C1366T/ C1367T/A1368G AHB0 protein: Q89H/L156F/T164I/T187I/Y204C/N226I/M285L/K324I/ 6.32 18 N326C/K381R gene: A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB0 protein: T187I/N226I/M321I/N326C/I406L/R426C 26.72 19 gene: C560T/G561T/A677T/C678T/G963T/A976T/A977G/T978C/ A1216T/T1218G/C1276T/T1278C AHB0 protein: V75E/L156F/T187I/N226I/L258I/M285L/N326C 36.12 20 gene: T224A/A468T/C560T/G561T/A677T/C678T/T772A/A774T/ A853C/A976T/A977G/T978C AHB0 protein: V75E/Q89H/L156F/T164I/T187I/N226I/N326C/K389I/ 7.49 21 I406L gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A677T/ C678T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G AHB0 protein: Q89H/T164I/T187 VY204C/N226I/K324I/N326C/K381R/ 26.81 22 R426C gene: A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/ C678T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB0 protein: D20V/Q89H/L156F/T187I/Y204C/M285L/K324I/N326C/K381R/ 47.97 23 K389I gene: A59T/C60G/A267T/A468T/C560T/G561T/A611G/T612C/ A853C/A971T/A972T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1166T/A1167C AHB0 protein: D20V/T164I/T187I/N226I/L258I/M285L/K324I/N326C 43.46 24 gene: A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/ T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C AHB0 protein: V75E/Q89H/T187I/Y204C/L258I/M321I/N326C/K381R/ 39.17 25 K389I/I406L/R426C gene: T224A/A267T/C560T/G561T/A611G/T612C/T772A/A774T/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB0 protein: V75E/Q89H/T164I/T187I/Y204C/N226I/M285L/M321I/ 44.00 26 N326C/K389I/R426C gene: T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB0 protein: D20V/V75E/T187I/N226I/L258I/M285L/N326C/R426C 31.40 27 gene: A59T/C60G/T224A/C560T/G561T/A677T/C678T/T772A/ A774T/A853C/A976T/A977G/T978C/C1276T/T1278C AHB0 protein: D20V/Q89H/L156F/T187I/N226I/N326C/K381R/K389I 30.12 28 gene: A59T/C60G/A267T/A468T/C560T/G561T/A677T/C678T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C AHB0 protein: D20V/V75E/L156F/T187I/N226I/M321I/K324I/N326C/ 38.56 29 R426C/P456L gene: A59T/C60G/T224A/A468T/C560T/G561T/A677T/C678T/ G963T/A971T/A972T/A976T/A977G/T978C/C1276T/ T1278C/C1366T/C1367T/A1368G AHB0 protein: V75E/Q89H/T164I/T187I/M321I/N326C/K381R/K389I/R426C 28.92 30 gene: T224A/A267T/C491T/A492T/C560T/G561T/G963T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/ C1276T/T1278C AHB0 protein: D20V/V75E/T164I/T187I/Y204C/M285L/M321I/N326C/ 16.78 31 K381R/R426C gene: A59T/C60G/T224A/C491T/A492T/C560T/G561T/A611G/ T612C/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB0 protein: V75E/T164I/T187I/M321I/N326C/K381R/I406L 27.79 32 gene: T224A/C491T/A492T/C560T/G561T/G963T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/A1216T/T1218G AHB0 protein: V75E/L156F/T187I/Y204C/M321I/N326C/K389I/R426C 46.48 33 gene: T224A/A468T/C560T/G561T/A611G/T612C/G963T/A976T/ A977G/T978C/A1166T/A1167C/C1276T/T1278C AHB0 protein: T187I/M285L/N326C/I406L/R426C/P456L 26.52 34 gene: C560T/G561T/A853C/A976T/A977G/T978C/A1216T/T1218G/ C1276T/T1278C/C1366T/C1367T/A1368G AHB0 protein: D20V/Q89H/T164I/T187I/M285L/N326C/K381R 37.80 35 gene: A59T/C60G/A267T/C491T/A492T/C560T/G561T/A853C/ A976T/A977G/T978C/A1141C/A1142G/A1143T AHB0 protein: Q89H/T187I/Y204C/L258I/M321I/K324I/N326C/K381R/ 7.01 36 R426C gene: A267T/C560T/G561T/A611G/T612C/T772A/A774T/G963T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB0 protein: V75E/Q89H/T187I/Y204C/L258I/M285L/M321I/N326C/ 26.53 37 I406L/R426C gene: T224A/A267T/C560T/G561T/A611G/T612C/T772A/A774T/ A853C/G963T/A976T/A977G/T978C/A1216T/T1218G/ C1276T/T1278C AHB0 protein: D20V/T187I/Y204C/L258I/N326C/K381R 24.57 38 gene: A59T/C60G/C560T/G561T/A611G/T612C/T772A/A774T/ A976T/A977G/T978C/A1141C/A1142G/A1143T AHB0 protein: T187I/N226I/N326C/I406L/P456L 10.62 39 gene: C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1216T/ T1218G/C1366T/C1367T/A1368G AHB0 protein: D20V/L156F/T187I/L258I/M321I/K324I/N326C 39.70 40 gene: A59T/C60G/A468T/C560T/G561T/T772A/A774T/G963T/ A971T/A972T/A976T/A977G/T978C AHB0 protein: V75E/Q89H/L156F/T187I/M321I/N326C/R426C 10.19 41 gene: T224A/A267T/A468T/C560T/G561T/G963T/A976T/A977G/ T978C/C1276T/T1278C AHB0 protein: T187I/Y204C/N226I/M285L/M321I/N326C/K381R/K389I/ 31.57 42 I406L/P456L gene: C560T/G561T/A611G/T612C/A677T/C678T/A853C/G963T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C/A1216T/T1218G/C1366T/C1367T/A1368G AHB0 protein: Q89H/T187I/N226I/M285L/M321I/K324I/N326C/K389I 38.43 43 gene: A267T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C/A1166T/A1167C AHB0 protein: V75E/L156F/T164I/T187I/Y204C/N226I/L258I/N326C/ 28.65 44 I406L gene: T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/T772A/A774T/A976T/A977G/T978C/A1216T/ T1218G AHB0 protein: V75E/Q89H/L156F/T164I/T187I/Y204C/M285L/N326C/ 40.19 45 K381R/K389I/R426C gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/ T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1276T/T1278C AHB0 protein: D20V/V75E/L156F/T187I/N326C/K381R/R426C 32.80 46 gene: A59T/C60G/T224A/A468T/C560T/G561T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB0 protein: V75E/T187I/L258I/N326C/K381R 30.10 47 gene: T224A/C560T/G561T/T772A/A774T/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB0 protein: D20V/V75E/T164I/T187I/M285L/M321I/N326C/K381R/ 15.71 48 R426C gene: A59T/C60G/T224A/C491T/A492T/C560T/G561T/A853C/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ C1276T/T1278C AHB0 protein: T187I/N226I/L258I/N326C/I406L 23.15 49 gene: C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/ T978C/A1216T/T1218G AHB0 protein: V75E/T164I/T187I/M321I/N326C/I406L 18.38 50 gene: T224A/C491T/A492T/C560T/G561T/G963T/A976T/A977G/ T978C/A1216T/T1218G AHB0 protein: T187I/L258I/M285L/M321I/N326C 40.49 51 gene: C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/ T978C AHB0 protein: D20V/Q89H/T187I/Y204C/L258I/M285L/N326C/K389I 29.46 52 gene: A59T/C60G/A267T/C560T/G561T/A611G/T612C/T772A/ A774T/A853C/A976T/A977G/T978C/A1166T/A1167C AHB0 protein: D20V/T187I/L258I/K324I/N326C/R426C 18.66 53 gene: A59T/C60G/C560T/G561T/T772A/A774T/A971T/A972T/ A976T/A977G/T978C/C1276T/T1278C AHB0 protein: T187I/Y204C/M285L/N326C/K381R 11.17 54 gene: C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB0 protein: T187I/Y204C/L258I/M285L/M321I/K324I/N326C/K381R/ 47.71 55 K389I/I406L gene: C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/A1216T/T1218G AHB0 protein: Q89H/T187I/L258I/M285L/K324I/N326C/I406L 24.72 56 gene: A267T/C560T/G561T/T772A/A774T/A853C/A971T/A972T/ A976T/A977G/T978C/A1216T/T1218G AHB0 protein: D20V/V75E/T187I/N226I/L258I/K324I/N326C/K381R/ 6.24 57 K389I/P456L gene: A59T/C60G/T224A/C560T/G561T/A677T/C678T/T772A/ A774T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB0 protein: D20V/T164I/T187I/N226I/L258I/N326C/K389I 33.98 58 gene: A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/ T772A/A774T/A976T/A977G/T978C/A1166T/A1167C AHB0 protein: T187I/Y204C/N326C/I406L/P456L 30.76 59 gene: C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1216T/ T1218G/C1366T/C1367T/A1368G AHB0 protein: D20V/T164I/T187I/L258I/M285L/K324I/N326C/I406L 11.13 60 gene: A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/ A853C/A971T/A972T/A976T/A977G/T978C/A1216T/T1218G AHB0 protein: Q89H/T187I/M285L/N326C/K381R 30.26 61 gene: A267T/C560T/G561T/A853C/A976T/A977G/T978C/A1141C/ A1142G/A1143T AHB0 protein: V75E/T187I/L258I/K324I/N326C/K389I/R426C/P456L 15.54 62 gene: T224A/C560T/G561T/T772A/A774T/A971T/A972T/A976T/ A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/ C1367T/A1368G AHB0 protein: D20V/Q89H/L156F/T187I/N226I/M321I/N326C/K381R/ 40.82 63 I406L gene: A59T/C60G/A267T/A468T/C560T/G561T/A677T/C678T/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1216T/T1218G AHB0 protein: V75E/T164I/T187I/N226I/M285L/K324I/N326C/K381R 47.01 64 gene: T224A/C491T/A492T/C560T/G561T/A677T/C678T/A853C/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T AHB0 protein: V75E/L156F/T164I/T187I/L258I/M285L/M321I/N326C/ 37.95 65 K381R/K389I/R426C gene: T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/ A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1276T/T1278C AHB0 protein: V75E/T164I/T187I/L258I/M2 85L/M321I/N326C/K381R/ 34.24 66 K389I/I406L/R426C gene: T224A/C491T/A492T/C560T/G561T/T772A/A774T/A853C/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB0 protein: D20V/V75E/Q89H/T187FL258I/M285L/M321I/N326C/ 39.39 67 K389I gene: A59T/C60G/T224A/A267T/C560T/G561T/T772A/A774T/ A853C/G963T/A976T/A977G/T978C/A1166T/A1167C AHB0 protein: T187I/N326C/K381R/K389I/P456L 19.34 68 gene: C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB0 protein: T164I/T187I/N226I/M285L/N326C/R426C 27.35 69 gene: C491T/A492T/C560T/G561T/A677T/C678T/A853C/A976T/ A977G/T978C/C1276T/T1278C AHB0 protein: D20V/V75E/Q89H/L156F/T187I/L258I/M285L/K324I/ 19.80 70 N326C gene: A59T/C60G/T224A/A267T/A468T/C560T/G561T/T772A/ A774T/A853C/A971T/A972T/A976T/A977G/T978C AHB0 protein: D20V/V75E/Q89H/T164I/T187I/Y204C/L258I/M285L/ 17.80 71 K324I/N326C gene: A59T/C60G/T224A/A267T/C491T/A492T/C560T/G561T/ A611G/T612C/T772A/A774T/A853C/A971T/A972T/A976T/ A977G/T978C AHB0 protein: L156F/T164I/T187I/K324I/N326C/R426C 33.38 72 gene: A468T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/ A977G/T978C/C1276T/T1278C AHB0 protein: D20V/V75E/L156F/T164I/T187I/L258I/M321I/K324I/N326C/ 14.46 73 K381R/I406L gene: A59T/C60G/T224A/A468T/C491T/A492T/C560T/G561T/ T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/A1216T/T1218G AHB0 protein: D20V/T187I/N226I/L258I/N326C/I406L 36.54 74 gene: A59T/C60G/C560T/G561T/A677T/C678T/T772A/A774T/ A976T/A977G/T978C/A1216T/T1218G AHB0 protein: V75E/Q89H/L156F/T187I/M321I/K324I/N326C 18.52 75 gene: T224A/A267T/A468T/C560T/G561T/G963T/A971T/A972T/ A976T/A977G/T978C AHB0 protein: L156F/T164I/T187I/K324I/N326C/K381R 11.35 76 gene: A468T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/ A977G/T978C/A1141C/A1142G/A1143T AHB0 protein: L156F/T164I/T187I/L258I/K324I/N326C 35.52 77 gene: A468T/C491T/A492T/C560T/G561T/T772A/A774T/A971T/ A972T/A976T/A977G/T978C AHB0 protein: D20V/V75E/Q89H/L156F/T187I/Y204C/L258I/M321I/ 34.31 78 N326C/K389I/I406L gene: A59T/C60G/T224A/A267T/A468T/C560T/G561T/A611G/ T612C/T772A/A774T/G963T/A976T/A977G/T978C/A1166T/ A1167C/A1216T/T1218G AHB0 protein: T187I/M285L/N326C/I406L/R426C 30.55 79 gene: C560T/G561T/A853C/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C AHB0 protein: T187I/N226I/N326C/K381R/P456L 22.81 80 gene: C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/C1366T/C1367T/A1368G AHB0 protein: D20V/T164I/T187I/Y204C/M285L/K324I/N326C/K389I/ 45.15 81 R426C gene: A59T/C60G/C491T/A492T/C560T/G561T/A611G/T612C/ A853C/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/ C1276T/T1278C AHB0 protein: V75E/T187I/N326C/K381R/I406L 27.19 82 gene: T224A/C560T/G561T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1216T/T1218G AHB0 protein: T187I/Y204C/N326C/K381R/K389I 20.17 83 gene: C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1166T/A1167C AHB0 protein: T187I/N226I/L258I/M285L/M321I/N326C 10.19 84 gene: C560T/G561T/A677T/C678T/T772A/A774T/A853C/ G963T/A976T/A977G/T978C AHB0 protein: D20V/L156F/T187I/M285L/M321I/K324I/N326C/K389I 49.44 85 gene: A59T/C60G/A468T/C560T/G561T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C/A1166T/A1167C AHB0 protein: D20V/L156F/T187I/Y204C/N326C/K389I/I406L/R426C 54.78 86 gene: A59T/C60G/A468T/C560T/G561T/A611G/T612C/A976T/ A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/ T1278C AHB0 protein: T164I/T187I/K324I/N326C/K389I/R426C 33.11 87 gene: C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/ T978C/A1166T/A1167C/C1276T/T1278C AHB0 protein: V75E/Q89H/T187I/M321I/K324I/N326C/I406L 27.31 88 gene: T224A/A267T/C560T/G561T/G963T/A971T/A972T/A976T/ A977G/T978C/A1216T/T1218G AHB0 protein: V75E/T187I/L258I/N326C/K389I 42.27 89 gene: T224A/C560T/G561T/T772A/A774T/A976T/A977G/T978C/ A1166T/A1167C AHB0 protein: D20V/V75E/L156F/T187I/N326C/K389I/I406L/R426C 47.72 90 gene: A59T/C60G/T224A/A468T/C560T/G561T/A976T/A977G/ T978C/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB0 protein: D20V/Q89H/T164I/T187I/N226I/N326C/K389I/I406L 16.27 91 gene: A59T/C60G/A267T/C491T/A492T/C560T/G561T/A677T/ C678T/A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G AHB0 protein: D20V/Q89H/L156F/T164I/T187I/L258I/M285L/N326C/ 37.40 92 K381R/R426C gene: A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/ T772A/A774T/A853C/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB0 protein: V75E/T164I/T187I/N226I/K324I/N326C/I406L 21.06 93 T224A/C491T/A492T/C560T/G561T/A677T/C678T/A971T/ A972T/A976T/A977G/T978C/A1216T/T1218G AHB0 protein: D20V/T164ET187I/L258I/M321I/N326C/K389I 34.04 94 A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/ G963T/A976T/A977G/T978C/A1166T/A1167C AHB0 protein: T187I/N226I/M285L/N326C/K381R/K389I 27.16 95 C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/ A1141C/A1142G/A1143T/A1166T/A1167C AHB0 protein: T187I/N226I/L258I/M285L/M321I/K324I/N326C/K381R/ 21.09 96 K389I/P456L gene: C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB0 protein: V75E/Q89H/L156F/T187I/M321I/N326C/I406L 13.79 97 gene: T224A/A267T/A468T/C560T/G561T/G963T/A976T/A977G/ T978C/A1216T/T1218G AHB0 protein: D20V/Q89H/L156F/T164I/T187I/N226I/M321I/N326C/ 15.28 98 K389I/I406L/R426C gene: A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/ A677T/C678T/G963T/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G/C1276T/T1278C AHB0 protein: V75E/T187I/Y204C/M285L/M321I/N326C/K381R/I406L/ 12.73 99 P456L gene: T224A/C560T/G561T/A611G/T612C/A853C/G963T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/ C1366T/C1367T/A1368G AHB1 protein: Q89H/T164I/T187I/M285L/M321I/N326C/K381R/K389I 30.85 00 gene: A267T/C491T/A492T/C560T/G561T/A853C/G963T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C AHB1 protein: T187I/N226I/L258I/M285L/M321I/N326C/K381R/I406L/ 47.93 01 R426C/P456L gene: C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/ C1276T/T1278C/C1366T/C1367T/A1368G AHB1 protein: L156F/T187I/Y204C/M321I/K324I/N326C 26.75 02 gene: A468T/C560T/G561T/A611G/T612C/G963T/A971T/A972T/ A976T/A977G/T978C AHB1 protein: D20V/L156F/T164I/T187I/M321I/K324I/N326C 22.66 03 gene: A59T/C60G/A468T/C491T/A492T/C560T/G561T/G963T/ A971T/A972T/A976T/A977G/T978C AHB1 protein: T187I/Y204C/L258I/K324I/N326C 18.88 04 gene: C560T/G561T/A611G/T612C/T772A/A774T/A971T/A972T/ A976T/A977G/T978C AHB1 protein: T164I/T187I/N226I/M285L/M321I/K324I/N326C/K381R/ 32.02 05 I406L/R426C gene: C491T/A492T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1216T/T1218G/C1276T/T1278C AHB1 protein: D20V/T164I/T187I/Y204C/N226I/N326C/K389I/I406L 27.92 06 gene: A59T/C60G/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G AHB1 protein: L156F/T187I/Y204C/N226I/M285L/M321I/N326C/I406L/ 26.52 07 R426C/P456L gene: A468T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/ G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/ T1278C/C1366T/C1367T/A1368G AHB1 protein: V75E/Q89H/L156F/T187I/K324I/N326C/K389I/R426C/P456L 35.35 08 gene: T224A/A267T/A468T/C560T/G5 61T/A971T/A972T/A976T/ A977G/T978C/A1166T/A1167C/C1276T/T1278C/C1366T/ C1367T/A1368G AHB1 protein: L156F/T164I/T187I/K324I/N326C/K381R/I406L/R426C 23.80 09 gene: A468T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/ C1276T/T1278C AHB1 protein: T164I/T187I/M321I/N326C/I406L 44.58 10 gene: C491T/A492T/C560T/G561T/G963T/A976T/A977G/T978C/ A1216T/T1218G AHB1 protein: D20V/Q89H/L156F/T164I/T187I/Y204C/M285L/K324I/ 34.80 11 N326C/K381R/R426C gene: A59T/C60G/A267T/A468T/C491T/A492T/C560T/G561T/ A611G/T612C/A853C/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB1 protein: L156F/T187I/Y204C/N326C/R426C 44.44 12 gene: A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/ C1276T/T1278C AHB1 protein: D20V/T164I/T187I/N226I/L258I/N326C/R426C 18.08 13 gene: A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/ T772A/A774T/A976T/A977G/T978C/C1276T/T1278C AHB1 protein: T187I/Y204C/N226I/L258I/M285L/M321I/N326C/K389I/ 14.79 14 I406L/R426C gene: C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/ A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G/C1276T/T1278C AHB1 protein: D20V/T187I/N226I/N326C/K389I 41.16 15 gene: A59T/C60G/C560T/G561T/A677T/C678T/A976T/A977G/ T978C/A1166T/A1167C AHB1 protein: V75E/Q89H/L156F/T187I/L258I/M321I/N326C/I406L/ 20.58 16 R426C gene: T224A/A267T/A468T/C560T/G561T/T772A/A774T/G963T/ A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C AHB1 protein: T187I/Y204C/N226I/M285L/N326C/R426C 52.49 17 gene: C560T/G561T/A611G/T612C/A677T/C678T/A853C/A976T/ A977G/T978C/C1276T/T1278C AHB1 protein: T187I/Y204C/M285L/N326C/K381R/R426C 23.80 18 gene: C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB1 protein: L156F/T187I/N226I/N326C/K381R 10.77 19 gene: A468T/C560T/G561T/A677T/C678T/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB1 protein: D20V/Q89H/T187I/Y204C/M285L/N326C/R426C 53.10 20 gene: A59T/C60G/A267T/C560T/G561T/A611G/T612C/A853C/ A976T/A977G/T978C/C1276T/T1278C AHB1 protein: L156F/T187I/L258I/M285L/M321I/K324I/N326C/K381R/ 18.82 21 I406L/R426C gene: A468T/C560T/G561T/T772A/A774T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1216T/T1218G/C1276T/T1278C AHB1 protein: T187I/Y204C/L258I/M285L/K324I/N326C 37.18 22 gene: C560T/G561T/A611G/T612C/T772A/A774T/A853C/A971T/ A972T/A976T/A977G/T978C AHB1 protein: D20V/T187I/Y204C/N226I/L258I/N326C/I406L/R426C 43.79 23 gene: A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/ T772A/A774T/A976T/A977G/T978C/A1216T/T1218G/C1276T/ T1278C AHB1 protein: D20V/L156F/T187I/N226I/M285L/N326C/K381R 25.49 24 gene: A59T/C60G/A468T/C560T/G561T/A677T/C678T/A853C/ A976T/A977G/T978C/A1141C/A1142G/A1143T AHB1 protein: D20V/Q89H/L156F/T187I/M285L/K324I/N326C/K389I 14.69 25 gene: A59T/C60G/A267T/A468T/C560T/G561T/A853C/A971T/ A972T/A976T/A977G/T978C/A1166T/A1167C AHB1 protein: V75E/T187I/K324I/N326C/K389I 39.72 26 gene: T224A/C560T/G561T/A971T/A972T/A976T/A977G/T978C/ A1166T/A1167C AHB1 protein: Q89H/L156F/T164I/T187I/Y204C/N226I/L258I/M285L/ 11.27 27 N326C/K389I/R426C gene: A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/ A1166T/A1167C/C1276T/T1278C AHB1 protein: Q89H/T187I/N226I/L258I/M321I/N326C/K381R/K389I/P456L 39.78 28 gene: A267T/C560T/G561T/A677T/C678T/T772A/A774T/G963T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A116617 A1167C/C1366T/C1367T/A1368G AHB1 protein: D20V/V75E/Q89H/T187I/Y204C/M321I/K324I/N326C/ 39.30 29 K381R gene: A59T/C60G/T224A/A267T/C560T/G561T/A611G/T612C/ G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T AHB1 protein: Q89H/T164I/T187I/M285L/N326C 34.10 30 gene: A267T/C491T/A492T/C560T/G561T/A853C/A976T/A977G/ T978C AHB1 protein: T187I/L258I/N326C/K381R/R426C 22.14 31 gene: C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/C1276T/T1278C AHB1 protein: L156F/T164I/T187I/N326C/K389I 21.70 32 gene: A468T/C491T/A492T/C560T/G561T/A976T/A977G/T978C/ A1166T/A1167C AHB1 protein: L156F/T164I/T187I/N226I/N326C/R426C 51.65 33 gene: A468T/C491T/A492T/C560T/G561T/A677T/C678T/A976T/ A977G/T978C/C1276T/T1278C AHB1 protein: D20V/Q89H/T187I/Y204C/M285L/M321I/N326C/K381R/R426C 52.00 34 gene: A59T/C60G/A267T/C560T/G561T/A611G/T612C/A853C/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ C1276T/T1278C AHB1 protein: V75E/Q89H/T187I/M321I/N326C/K381R/K389I/I406L/P456L 24.62 35 gene: T224A/A267T/C560T/G561T/G963T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/T1218G/ C1366T/C1367T/A1368G AHB1 protein: D20V/T164I/T187I/M321I/N326C/K389I/P456L 43.89 36 gene: A59T/C60G/C491T/A492T/C560T/G561T/G963T/A976T/ A977G/T978C/A1166T/A1167C/C1366T/C1367T/A1368G AHB1 protein: Q89H/T187I/L258I/N326C/P456L 42.33 37 gene: A267T/C560T/G561T/T772A/A774T/A976T/A977G/T978C/ C1366T/C1367T/A1368G AHB1 protein: V75E/L156F/T164I/T187I/L258I/M285L/N326C/K381R/ 28.66 38 K389FR426C gene: T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/ A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/C1276T/T1278C AHB1 protein: T187I/N226I/M285L/K324I/N326C 18.06 39 gene: C560T/G561T/A677T/C678T/A853C/A971T/A972T/A976T/ A977G/T978C AHB1 protein: T187I/L258I/N326C/K381R/K389I/I406L 14.27 40 gene: C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1166T/A1167C/A1216T/T1218G AHB1 protein: D20V/T187I/L258I/K324I/N326C/I406L 30.60 41 gene: A59T/C60G/C560T/G561T/T772A/A774T/A971T/A972T/ A976T/A977G/T978C/A1216T/T1218G AHB1 protein: V75E/L156F/T164I/T187I/Y204C/L258I/N326C/K389I/ 37.56 42 I406L gene: T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G AHB1 protein: D20V/Q89H/L156F/T187I/K324I/N326C/K381R/R426C 14.03 43 gene: A59T/C60G/A267T/A468T/C560T/G561T/A971T/A972T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/ T1278C AHB1 protein: Q89H/T187I/N226I/M285L/M321I/K324I/N326C 10.59 44 gene: A267T/C560T/G561T/A677T/C678T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C AHB1 protein: T164I/T187I/M285L/M321I/N326C/K381R 11.91 45 gene: C491T/A492T/C560T/G561T/A853C/G963T/A976T/A977G/ T978C/A1141C/A1142G/A1143T AHB1 protein: Q89H/L156F/T187I/Y204C/N226I/M285L/M321I/N326C/ 34.37 46 K389I/R426C gene: A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/ A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/ C1276T/T1278C AHB1 protein: Q89H/L156F/T164I/T187I/L258I/N326C/K389I 34.78 47 gene: A267T/A468T/C491T/A492T/C560T/G561T/T772A/A774T/ A976T/A977G/T978C/A1166T/A1167C AHB1 protein: T164I/T187I/N226I/M321I/K324I/N326C/K381R 29.96 48 gene: C491T/A492T/C560T/G561T/A677T/C678T/G963T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T AHB1 protein: D20V/Q89H/T164I/T187I/Y204C/N226I/M285L/N326C/ 47.59 49 K381R/K389I/I406L gene: A59T/C60G/A267T/C491T/A492T/C560T/G561T/A611G/ T612C/A677T/C678T/A853C/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1166T/A1167C/A1216T/T1218G AHB1 protein: V75E/T164I/T187I/L258I/N326C/K389I 26.46 50 gene: T224A/C491T/A492T/C560T/G561T/T772A/A774T/A976T/ A977G/T978C/A1166T/A1167C AHB1 protein: T187I/N326C/K389I/I406L/R426C/P456L 29.62 51 gene: C560T/G561T/A976T/A977G/T978C/A1166T/A1167C/A1216T/ T1218G/C1276T/T1278C/C1366T/C1367T/A1368G AHB1 protein: V75E/T187I/N326C/K381R/K389I/I406L/R426C 15.74 52 gene: T224A/C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/ T1278C AHB1 protein: T187I/L258I/M321I/N326C/I406L 36.73 53 gene: C560T/G561T/T772A/A774T/G963T/A976T/A977G/T978C/ A1216T/T1218G AHB1 protein: Q89H/L156F/T164I/T187I/Y204C/N226I/K324I/N326C/ 47.99 54 P456L gene: A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/A971T/A972T/A976T/A977G/T978C/C1366T/ C1367T/A1368G AHB1 protein: T187I/N226I/L258I/M285L/M321I/K324I/N326C/I406L/ 29.70 55 R426C/P456L gene: C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C/A1216T/T1218G/ C1276T/T1278C/C1366T/C1367T/A1368G AHB1 protein: D20V/T187I/M285L/N326C/K381R 49.63 56 gene: A59T/C60G/C560T/G561T/A853C/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB1 protein: V75E/T187I/Y204C/M321I/N326C 39.42 57 gene: T224A/C560T/G561T/A611G/T612C/G963T/A976T/A977G/ T978C AHB1 protein: T164I/T187I/Y204C/L258I/K324I/N326C/K389I 40.96 58 gene: C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/ A971T/A972T/A976T/A977G/T978C/A1166T/A1167C AHB1 protein: V75E/L156F/T164I/T187I/Y204C/L258I/M285L/M321I/N326C/ 49.07 59 K389I/I406L gene: T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ T772A/A774T/A853C/G963T/A976T/A977G/T978C/A1166T/ A1167C/A1216T/T1218G AHB1 protein: V75E/T164I/T187I/L258I/M285L/M321I/K324I/N326C/I406L/ 39.24 60 R426C gene: T224A/C491T/A492T/C560T/G561T/T772A/A774T/A853C/ G963T/A971T/A972T/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C AHB1 protein: D20V/T187I/M285L/N326C/K389I/I406L/R426C 25.46 61 gene: A59T/C60G/C560T/G561T/A853C/A976T/A977G/T978C/ A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB1 protein: T187I/M285L/M321I/N326C/R426C 37.91 62 gene: C560T/G561T/A853C/G963T/A976T/A977G/T978C/C1276T/ T1278C AHB1 protein: Q89H/L156F/T164I/T187I/N226I/L258I/M321I/K324I/N326C 47.32 63 gene: A267T/A468T/C491T/A492T/C560T/G561T/A677T/C678T/ T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C AHB1 protein: Q89H/T164I/T187I/Y204C/L258I/N326C 40.15 64 gene: A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/ A774T/A976T/A977G/T978C AHB1 protein: T187I/M285L/M321I/N326C/I406L/R426C 42.51 65 gene: C560T/G561T/A853C/G963T/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C AHB1 protein: D20V/L156F/T187I/M285L/M321I/K324I/N326C/R426C 17.90 66 gene: A59T/C60G/A468T/C560T/G561T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C/C1276T/T1278C AHB1 protein: D20V/T187I/M285L/M321I/K324I/N326C/K389I 14.86 67 gene: A59T/C60G/C560T/G561T/A853C/G963T/A971T/A972T/ A976T/A977G/T978C/A1166T/A1167C AHB1 protein: L156F/T164I/T187I/L258I/K324I/N326C/K389I 15.42 68 gene: A468T/C491T/A492T/C560T/G561T/T772A/A774T/A971T/ A972T/A976T/A977G/T978C/A1166T/A1167C AHB1 protein: T187I/M285L/N326C/K381R/I406L/R426C 34.56 69 gene: C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1216T/T1218G/C1276T/T1278C AHB1 protein: V75E/Q89H/L156F/T164I/T187I/Y204C/N226I/M285L/ 35.63 70 K324I/N326C gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/ T612C/A677T/C678T/A853C/A971T/A972T/A976T/A977G/ T978C AHB1 protein: V75E/L156F/T164I/T187I/Y204C/M285L/N326C 34.13 71 gene: T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A853C/A976T/A977G/T978C AHB1 protein: D20V/T187I/M285L/M321I/K324I/N326C/R426C 33.60 72 gene: A59T/C60G/C560T/G561T/A853C/G963T/A971T/A972T/ A976T/A977G/T978C/C1276T/T1278C AHB1 protein: T187I/Y204C/K324I/N326C/I406L/R426C 15.43 73 gene: C560T/G561T/A611G/T612C/A971T/A972T/A976T/A977G/ T978C/A1216T/T1218G/C1276T/T1278C AHB1 protein: V75E/Q89H/L156F/T187EY204C/N326C 43.57 74 gene: T224A/A267T/A468T/C560T/G561T/A611G/T612C/A976T/ A977G/T978C AHB1 protein: Q89H/T164I/T187I/M285L/M321I/K324I/N326C/K381R/ 13.09 75 K389I/I406L/P456L gene: A267T/C491T/A492T/C560T/G561T/A853C/G963T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G AHB1 protein: T164I/T187I/L258I/M285L/M321I/N326C/K381R/K389I/ 48.38 76 I406L/P456L gene: C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C/A1216T/T1218G/C1366T/C1367T/A1368G AHB1 protein: T187I/L258I/N326C/I406L/R426C 41.69 77 gene: C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C AHB1 protein: V75E/T164I/T187I/L258I/N326C/K381R/R426C 44.62 78 gene: T224A/C491T/A492T/C560T/G561T/T772A/A774T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB1 protein: D20V/T187I/N226I/M285L/N326C/R426C 52.03 79 gene: A59T/C60G/C560T/G561T/A677T/C678T/A853C/A976T/ A977G/T978C/C1276T/T1278C AHB1 protein: V75E/Q89H/T164I/T187I/Y204C/L258I/N326C/I406L 30.42 80 gene: T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/ T772A/A774T/A976T/A977G/T978C/A1216T/T1218G AHB1 protein: L156F/T187I/M321I/N326C/I406L 34.17 81 gene: A468T/C560T/G561T/G963T/A976T/A977G/T978C/A1216T/ T1218G AHB1 protein: V75E/Q89H/L156F/T164I/T187I/Y204C/L258I/M285L/ 51.41 82 M321I/N326C/K389I gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611G/ T612C/T772A/A774T/A853C/G963T/A976T/A977G/T978C/ A1166T/A1167C AHB1 protein: V75E/T164I/T187I/N226I/L258I/M285L/K324I/N326C/ 42.59 83 K389I gene: T224A/C491T/A492T/C560T/G561T/A677T/C678T/T772A/ A774T/A853C/A971T/A972T/A976T/A977G/T978C/A1166T/ A1167C AHB1 protein: V75E/T187I/M285L/K324I/N326C/R426C 15.71 84 gene: T224A/C560T/G561T/A853C/A971T/A972T/A976T/A977G/ T978C/C1276T/T1278C AHB1 protein: D20V/Q89H/T164I/T187I/N226I/L258I/M321I/K324I/N326C/ 24.71 85 K381R/P456L gene: A59T/C60G/A267T/C491T/A492T/C560T/G561T/A677T/ C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/C1366T/C1367T/A1368G AHB1 protein: V75E/Q89H/T164I/T187I/Y204C/L258I/M321I/N326C 33.23 86 gene: T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/ T772A/A774T/G963T/A976T/A977G/T978C AHB1 protein: Q89H/L156F/T164I/T187I/Y204C/N226I/L258I/M285L/ 40.29 87 N326C/K389I gene: A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/T772A/A774T/A853C/A976T/A977G/T978C/ A1166T/A1167C AHB1 protein: Q89H/L156F/T187I/Y204C/N226I/M321I/K324I/N326C/ 25.52 88 K381R/I406L gene: A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/ G963T/A971T/A972T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1216T/T1218G AHB1 protein: L156F/T187I/M285L/N326C/R426C 32.17 89 gene: A468T/C560T/G561T/A853C/A976T/A977G/T978C/C1276T/ T1278C AHB1 protein: T187I/N226I/M285L/M321I/N326C/K381R/K389I/I406L/ 10.02 90 R426C/P456L gene: C560T/G561T/A677T/C678T/A853C/G963T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/A1166T/A1167C/A1216T/ T1218G/C1276T/T1278C/C1366T/C1367T/A1368G AHB1 protein: Q89H/T187I/N326C/R426C/P456L 19.01 91 gene: A267T/C560T/G561T/A976T/A977G/T978C/C1276T/T1278C/ C1366T/C1367T/A1368G AHB1 protein: Q89H/T187I/Y204C/K324I/N326C/K389I/I406L/R426C 36.57 92 gene: A267T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/ A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/ T1278C AHB1 protein: D20V/Q89H/T164I/T187I/N226I/M285L/M321I/N326C/ 11.61 93 K381R/P456L gene: A59T/C60G/A267T/C491T/A492T/C560T/G561T/A677T/ C678T/A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1366T/C1367T/A1368G AHB1 protein: V75E/L156F/T187I/Y204C/L258I/M321I/K324I/N326C/ 37.01 94 K381R/I406L/R426C gene: T224A/A468T/C560T/G561T/A611G/T612C/T772A/A774T/ G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1216T/T1218G/C1276T/T1278C AHB1 protein: D20V/T187I/N226I/N326C/K381R 12.80 95 gene: A59T/C60G/C560T/G561T/A677T/C678T/A976T/A977G/ T978C/A1141C/A1142G/A1143T AHB1 protein: D20 V/L156F/T187I/M285L/K324I/N326C/K381R/K389I 37.25 96 gene: A59T/C60G/A468T/C560T/G561T/A853C/A971T/A972T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C AHB1 protein: Q89H/L156F/T164I/T187I/Y204C/M285L/K324I/N326C/ 22.56 97 K381R gene: A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A853C/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T AHB1 protein: V75E/L156F/T187I/N326C/K389I/R426C 47.60 98 gene: T224A/A468T/C560T/G561T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB1 protein: T187I/Y204C/N226I/N326C/K381R 24.39 99 gene: C560T/G561T/A611G/T612C/A677T/C678T/A976T/A977G/ T978C/A1141C/A1142G/A1143T AHB2 protein: T187I/Y204C/L258I/M285L/M321I/K324I/N326C/K381R/ 31.79 00 I406L/R426C gene: C560T/G561T/A611G/T612C/T772A/A774T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1216T/T1218G/C1276T/T1278C AHB2 protein: T187I/L258I/K324I/N326C/K389I/I406L 22.84 01 gene: C560T/G561T/T772A/A774T/A971T/A972T/A976T/A977G/ T978C/A1166T/A1167C/A1216T/T1218G AHB2 protein: T187I/M321I/K324I/N326C/K381R 16.67 02 gene: C560T/G561T/G963T/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB2 protein: V75E/Q89H/T164I/T187I/Y204C/N326C/K389I 53.92 03 gene: T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/ A976T/A977G/T978C/A1166T/A1167C AHB2 protein: V75E/Q89H/T187I/N226I/N326C/K389I 30.13 04 gene: T224A/A267T/C560T/G561T/A677T/C678T/A976T/A977G/ T978C/A1166T/A1167C AHB2 protein: T187I/Y204C/M285L/N326C/P456L 35.97 05 gene: C560T/G561T/A611G/T612C/A853C/A976T/A977G/T978C/ C1366T/C1367T/A1368G AHB2 protein: L156F/T187I/Y204C/K324I/N326C/K381R/R426C 19.71 06 gene: A468T/C560T/G561T/A611G/T612C/A971T/A972T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB2 protein: Q89H/T164I/T187I/M321I/N326C/I406L 36.98 07 gene: A267T/C491T/A492T/C560T/G561T/G963T/A976T/A977G/ T978C/A1216T/T1218G AHB2 protein: T164I/T187I/L156F/K324I/N326C 26.84 08 gene: C491T/A492T/C560T/G561T/A468T/A971T/A972T/A976T/ A977G/T978C AHB2 protein: D20V/V75E/T187I/N226I/M321I/K324I/N326C 19.99 09 gene: A59T/C60G/T224A/C560T/G561T/A677T/C678T/G963T/ A971T/A972T/A976T/A977G/T978C AHB2 protein: T187I/K324I/N326C/I406L/R426C/P456L 17.98 10 gene: C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C/C1366T/C1367T/A1368G AHB2 protein: T164I/T187I/K324I/N326C/R426C 43.42 11 gene: C491T/A492T/C560T/G561T/A971T/A972T/A976T/A977G/ T978C/C1276T/T1278C AHB2 protein: T164I/T187I/L258I/M285L/M321I/K324I/N326C/K381R/ 40.53 12 K389I/P456L gene: C491T/A492T/C560T/G561T/T772A/A774T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB2 protein: Q89H/T164I/T187I/Y204C/N226I/L258I/M321I/N326C/R426C 43.62 13 gene: A267T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/ C678T/T772A/A774T/G963T/A976T/A977G/T978C/C1276T/ T1278C AHB2 protein: Q89H/T187I/N226I/M321I/N326C 33.19 14 gene: A267T/C560T/G561T/A677T/C678T/G963T/A976T/A977G/ T978C AHB2 protein: Q89H/L156F/T187I/Y204C/N326C/K381R/I406L/R426C/P456L 17.80 15 gene: A267T/A468T/C560T/G561T/A611G/T612C/A976T/A977G/ T978C/A1141C/A1142G/A1143T/A1216T/T1218G/C1276T/ T1278C/C1366T/C1367T/A1368G AHB2 protein: D20V/T187I/M321I/K324I/N326C/K381R/I406L/R426C 23.32 16 gene: A59T/C60G/C560T/G561T/G963T/A971T/A972T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1216T/T1218G/ C1276T/T1278C AHB2 protein: T187I/N226I/L258I/N326C/I406L/P456L 37.34 17 gene: C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/ T978C/A1216T/T1218G/C1366T/C1367T/A1368G AHB2 protein: L156F/T187I/L258I/M321I/N326C 39.18 18 gene: A468T/C560T/G561T/T772A/A774T/G963T/A976T/A977G/ T978C AHB2 protein: T164I/T187I/Y204C/N226I/L258I/M321I/N326C/K381R/ 10.89 19 K389I/I406L/P456L gene: C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/T772A/ A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/A1216T/T1218G/C1366T/C1367T/A1368G AHB2 protein: D20V/L156F/T187I/N226I/N326C 10.50 20 gene: A59T/C60G/A468T/C560T/G561T/A677T/C678T/A976T/ A977G/T978C AHB2 protein: V75E/Q89H/L156F/T187I/N226I/M321I/K324I/N326C/ 43.86 21 R426C gene: T224A/A267T/A468T/C560T/G561T/A677T/C678T/G963T/ A971T/A972T/A976T/A977G/T978C/C1276T/T1278C AHB2 protein: D20V/T187I/Y204C/N226I/K324I/N326C/K381R/R426C 31.14 22 gene: A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB2 protein: Q89H/T164I/T187I/Y204C/L258I/M321I/K324I/N326C/ 43.83 23 K389I/I406L/R426C gene: A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/ A774T/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/ A1167C/A1216T/T1218G/C1276T/T1278C AHB2 protein: D20V/L156F/T187I/M285L/N326C 34.58 24 gene: A59T/C60G/A468T/C560T/G561T/A853C/A976T/A977G/ T978C AHB2 protein: V75E/T187I/Y204C/N226I/K324I/N326C/K381R/I406L 15.40 25 gene: T224A/C560T/G561T/A611G/T612C/A677T/C678T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1216T/T1218G AHB2 protein: V75E/T187I/M321I/I406L/N326C/R426C 46.83 26 gene: T224A/C560T/G561T/G963T/A1216T/T1218G/A976T/ A977G/T978C/C1276T/T1278C AHB2 protein: Q89H/T187I/L258I/N326C/R426C 24.10 27 gene: A267T/C560T/G561T/T772A/A774T/A976T/A977G/T978 C/C1276T/T1278C AHB2 protein: V75E/Q89H/L156F/T164FT187I/Y204C/M285L/M321I/ 40.78 28 N326C/K389I gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A611 G/T612C/A853C/G963T/A976T/A977G/T978C/A1166T/ A1167C AHB2 protein: L156F/T187I/Y204C/N226I/M285L/K324I/N326C/K389I/ 13.16 29 R426C/P456L gene: A468T/C560T/G561T/A611G/T612C/A677T/C678T/A853C/ A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/ C1276T/T1278C/C1366T/C1367T/A1368G AHB2 protein: T187I/N226I/M285L/N326C/R426C 50.93 30 gene: C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/ C1276T/T1278C AHB2 protein: V75E/Q89H/T164I/T187I/L258I/M285L/M321I/N326C/ 25.23 31 K389I/R426C gene: T224A/A267T/C491T/A492T/C560T/G561T/T772A/A774T/ A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/ C1276T/T1278C AHB2 protein: D20V/T187I/L258I/M321I/N326C/R426C 28.21 32 gene: A59T/C60G/C560T/G561T/T772A/A774T/G963T/A976T/ A977G/T978C/C1276T/T1278C AHB2 protein: D20V/L156F/T187I/Y204C/N226I/L258I/K324I/N326C/ 21.56 33 K381R/R426C gene: A59T/C60G/A468T/C560T/G561T/A611G/T612C/A677T/ C678T/T772A/A774T/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB2 protein: D20V/L156F/T187I/Y204C/K324I/N326C 39.25 34 gene: A59T/C60G/A468T/C560T/G561T/A611G/T612C/A971T/ A972T/A976T/A977G/T978C AHB2 protein: V75E/Q89H/T187I/N326C/K389I/R426C 13.50 35 gene: T224A/A267T/C560T/G561T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB2 protein: T164I/T187I/Y204C/L258I/M321I/K324I/N326C/K389I 24.76 36 gene: C491T/A492T/C560T/G561T/A611G/T612C/T772A/A774T/ G963T/A971T/A972T/A976T/A977G/T978C/A1166T/A1167C AHB2 protein: D20V/T164I/T187I/M285L/N326C 15.38 37 gene: A59T/C60G/C491T/A492T/C560T/G561T/A853C/A976T/ A977G/T978C AHB2 protein: T164I/T187I/Y204C/N226I/K324I/N326C/K389I/I406L 28.69 38 gene: C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/ A971T/A972T/A976T/A977G/T978C/A1166T/A1167C/A1216T/ T1218G AHB2 protein: T187I/L258I/M285L/M321I/K324I/N326C/K381R/I406L 12.32 39 gene: C560T/G561T/T772A/A774T/A853C/G963T/A971T/A972T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/ T1218G AHB2 protein: T187I/Y204C/N226I/L258I/M321I/N326C/I406L 32.95 40 gene: C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/ G963T/A976T/A977G/T978C/A1216T/T1218G AHB2 protein: V75E/T187I/N226I/N326C/K381R 11.20 41 gene: T224A/C560T/G561T/A677T/C678T/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB2 protein: V75E/T187I/Y204C/N326C/I406L 22.36 42 gene: T224A/C560T/G561T/A611G/T612C/A976T/A977G/T978C/ A1216T/T1218G AHB2 protein: V75E/T187I/Y204C/N226I/M285L/K324I/N326C/K381R/ 31.88 43 R426C gene: T224A/C560T/G561T/A611G/T612C/A677T/C678T/A853C/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB2 protein: Q89H/T187I/M285L/N326C/R426C 15.97 44 gene: A267T/C560T/G561T/A853C/A976T/A977G/T978C/C1276T/ T1278C AHB2 protein: Q89H/T187I/K324I/N326C/K389I 50.05 45 gene: A267T/C560T/G561T/A971T/A972T/A976T/A977G/T978C/ A1166T/A1167C AHB2 protein: Q89H/T164I/T187I/K324I/N326C/K389I 17.15 46 gene: A267T/C491T/A492T/C560T/G561T/A971T/A972T/A976T/ A977G/T978C/A1166T/A1167C AHB2 protein: L156F/T187I/Y204C/L25 8I/M285L/M321I/N326C/K381R/K389I 46.09 47 gene: A468T/C560T/G561T/A611G/T612C/T772A/A774T/A853C/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C AHB2 protein: T187I/K324I/N326C/K381R/K389I/I406L 27.96 48 gene: C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1166T/A1167C/A1216T/T1218G AHB2 protein: V75E/Q89H/T164I/T187VY204C/M285L/N326C/K389I/ 52.79 49 R426C gene: T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/ A853C/A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C AHB2 protein: V75E/T187I/Y204C/N226I/L258I/N326C/I406L 46.36 50 gene: T224A/C560T/G561T/A611G/T612C/A677T/C678T/T772A/ A774T/A976T/A977G/T978C/A1216T/T1218G AHB2 protein: T187I/Y204C/N226I/K324I/N326C/R426C 41.42 51 gene: C560T/G561T/A611G/T612C/A677T/C678T/A971T/A972T/ A976T/A977G/T978C/C1276T/T1278C AHB2 protein: V75E/Q89H/L156F/T164I/T187I/M285L/N326C/K381R/ 31.37 52 K389I/R426C gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A853C/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C/C1276T/T1278C AHB2 protein: D20V/L156F/T187I/M321I/N326C/I406L/R426C 17.17 53 gene: A59T/C60G/A468T/C560T/G561T/G963T/A976T/A977G/ T978C/A1216T/T1218G/C1276T/T1278C AHB2 protein: D20V/T187I/Y204C/N226I/M285L/K324I/N326C/R426C 18.13 54 gene: A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/ A853C/A971T/A972T/A976T/A977G/T978C/C1276T/ T1278C AHB2 protein: T164I/T187I/N226I/L258I/M285L/M321I/N326C/K381R/R426C 40.54 55 gene: C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/ A853C/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/C1276T/T1278C AHB2 protein: V75E/Q89H/L156F/T187I/N226I/M285L/N326C/K381R/ 27.05 56 K389I gene: T224A/A267T/A468T/C560T/G561T/A677T/C678T/A853C/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C AHB2 protein: D20V/T187I/N226I/M285L/K324I/N326C/K381R/R426C 26.95 57 gene: A59T/C60G/C560T/G561T/A677T/C678T/A853C/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ C1276T/T1278C AHB2 protein: T187I/Y204C/M321I/N326C/K381R 26.92 58 gene: C560T/G561T/A611G/T612C/G963T/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB2 protein: V75E/T164I/T187I/N226I/L258I/N326C/K389I 45.00 59 gene: T224A/C491T/A492T/C560T/G561T/A677T/C678T/T772A/ A774T/A976T/A977G/T978C/A1166T/A1167C AHB2 protein: D20V/L156F/T164I/T187I/L258I/N326C/K381R/K389I/ 40.38 60 I406L/R426C gene: A59T/C60G/A468T/C491T/A492T/C560T/G561T/T772A/ A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB2 protein: D20V/V75E/Q89H/L156F/T164I/T187I/L258I/N326C/ 38.67 61 K381R/K389I/I406L gene: A59T/C60G/T224A/A267T/A468T/C491T/A492T/C560T/ G561T/T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/A1216T/T1218G AHB2 protein: T187I/N226I/N326C/K381R/I406L/R426C 46.09 62 gene: C560T/G561T/A677T/C678T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1216T/T1218G/C1276T/T1278C AHB2 protein: Q89H/T187I/Y204C/N326C/I406L 16.60 63 gene: A267T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/ A1216T/T1218G AHB2 protein: D20V/L156F/T164I/T187I/Y204C/N226I/M321I/K324I/ 37.96 64 N326C/K389I/R426C gene: A59T/C60G/A468T/C491T/A492T/C560T/G561T/A611G/ T612C/A677T/C678T/G963T/A971T/A972T/A976T/A977G/ T978C/A1166T/A1167C/C1276T/T1278C AHB2 protein: D20V/T164I/T187I/L258I/K324I/N326C/K381R/I406L 10.91 65 gene: A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1216T/T1218G AHB2 protein: V75E/T164I/T187I/M321I/K324I/N326C/K389I/R426C/ 46.29 66 P456L gene: T224A/C491T/A492T/C560T/G561T/G963T/A971T/A972T/ A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/ C1366T/C1367T/A1368G AHB2 protein: Q89H/T187I/Y204C/N226I/N326C/K389I 16.72 67 gene: A267T/C560T/G561T/A611G/T612C/A677T/C678T/A976T/ A977G/T978C/A1166T/A1167C AHB2 protein: L156F/T187I/M321I/N326C/K389I/R426C 38.04 68 gene: A468T/C560T/G561T/G963T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB2 protein: Q89H/L156F/T187I/N226I/L258I/M285L/N326C/K389I/ 18.33 69 I406L/R426C gene: A267T/A468T/C560T/G561T/A677T/C678T/T772A/A774T/ A853C/A976T/A977G/T978C/A1166T/A1167C/A1216T/ T1218G/C1276T/T1278C AHB2 protein: Q89H/L156F/T187I/Y204C/M285L/M321I/N326C/R426C/ 44.17 70 P456L gene: A267T/A468T/C560T/G561T/A611G/T612C/A853C/G963T/ A976T/A977G/T978C/C1276T/T1278C/C1366T/C1367T/ A1368G AHB2 protein: Q89H/L156F/T187I/N226FK324I/N326C/K389I/I406L/R426C 71 gene: A267T/A468T/C560T/G561T/A677T/C678T/A971T/A972T/ 14.39 A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G/ C1276T/T1278C AHB2 protein: T164I/T187I/N226I/N326C/K381R 24.88 72 gene: C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/ T978C/A1141C/A1142G/A1143T AHB2 protein: V75E/Q89H/T187I/Y204C/N226I/L258I/K324I/N326C/K381R 73 gene: T224A/A267T/C560T/G561T/A611G/T612C/A677T/C678T/ 31.25 T772A/A774T/A971T/A972T/A976T/A977G/T978C/A1141C/ A1142G/A1143T AHB2 protein: Q89H/T164I/T187I/Y204C/M321FK324I/N326C/K381R/ 45.22 74 I406L/R426C gene: A267T/C491T/A492T/C560T/G561T/A611G/T612C/G963T/ A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1216T/T1218G/C1276T/T1278C AHB2 protein: D20V/V75E/Q89H/T164I/T187I/M285L/M321I/K324I/N326C/ 28.25 75 K381R gene: A59T/C60G/T224A/A267T/C491T/A492T/C560T/G561T/ A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/ A1142G/A1143T AHB2 protein: D20V/T187I/N326C/K381R/R426C 39.81 76 gene: A59T/C60G/C560T/G561T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/C1276T/T1278C AHB2 protein: T187I/K324I/N326C/K389I/R426C 40.72 77 gene: C560T/G561T/A971T/A972T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB2 protein: Q89H/T187I/L258I/M285L/N326C/K389I/I406L 21.71 78 gene: A267T/C560T/G561T/T772A/A774T/A853C/A976T/A977G/ T978C/A1166T/A1167C/A1216T/T1218G AHB2 protein: D20V/T187I/M321I/N326C/I406L 30.39 79 gene: A59T/C60G/C560T/G561T/G963T/A976T/A977G/T978C/ A1216T/T1218G AHB2 protein: V75E/Q89H/T187I/L258I/M321I/N326C/I406L/R426C 25.67 80 gene: T224A/A267T/C560T/G561T/T772A/A774T/G963T/A976T/ A977G/T978C/A1216T/T1218G/C1276T/T1278C AHB2 protein: T187I/N226I/L258I/M321I/N326C/R426C 23.08 81 gene: C560T/G561T/A677T/C678T/T772A/A774T/G963T/A976T/ A977G/T978C/C1276T/T1278C AHB2 protein: T164I/T187I/Y204C/N226I/L258I/M321I/K324I/N326C/ 45.52 82 K381R/R426C gene: C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/ T772A/A774T/G963T/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB2 protein: L156F/T164I/T187I/Y204C/N326C/K381R/R426C 37.98 83 gene: A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A976T/A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB2 protein: T187I/N226I/M285L/K324I/N326C/R426C 48.04 84 gene: C560T/G561T/A677T/C678T/A853C/A971T/A972T/A976T/ A977G/T978C/C1276T/T1278C AHB2 protein: D20V/T187I/Y204C/N226I/M321I/N326C/I406L/R426C 37.40 85 gene: A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/ G963T/A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C AHB2 protein: Q89H/L156F/T187I/Y204C/N226I/L258I/M285L/M321I/ 34.96 86 K324I/N326C gene: A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/ T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/ T978C AHB2 protein: V75E/L156F/T164I/T187I/L258I/N326C 32.41 87 gene: T224A/A468T/C491T/A492T/C560T/G561T/T772A/A774T/ A976T/A977G/T978C AHB2 protein: D20V/V75E/T164I/T187I/Y204C/N326C 19.27 88 gene: A59T/C60G/T224A/C491T/A492T/C560T/G561T/A611G/ T612C/A976T/A977G/T978C AHB2 protein: D20V/T187I/Y204C/K324I/N326C 26.57 89 gene: A59T/C60G/C5 60T/G561T/A611G/T612C/A971T/A972T/ A976T/A977G/T978C AHB2 protein: L156F/T187I/Y204C/N326C/K381R/R426C 23.85 90 gene: A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB2 protein: V75E/Q89H/T164I/T187I/N326C 41.43 91 gene: T224A/A267T/C491T/A492T/C560T/G561T/A976T/A977G/ T978C AHB2 protein: D20V/T164I/T187I/N226I/K324I/N326C 17.12 92 gene: A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/ A971T/A972T/A976T/A977G/T978C AHB2 protein: T187I/N226I/L258I/M285L/M321I/K324I/N326C 35.48 93 gene: C560T/G561T/A677T/C678T/T772A/A774T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C AHB2 protein: T187I/N226I/L258I/N326C/P456L 31.00 94 gene: C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/ T978C/C1366T/C1367T/A1368G AHB2 protein: T64I/T187I/N226I/K324I/N326C/K389I/P456L 53.54 95 gene: C491T/A492T/C560T/G561T/A677T/C678T/A971T/A972T/ A976T/A977G/T978C/A1166T/A1167C/C1366T/C1367T/ A1368G AHB2 protein: D20V/T187I/Y204C/L258I/M285L/M321I/K324I/N326C/ 13.89 96 K389I/R426C gene: A59T/C60G/C560T/G561T/A611G/T612C/T772A/A774T/ A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB2 protein: V75E/L156F/T187I/Y204C/L258I/M321I/N326C/K381R/ 23.26 97 I406L/R426C gene: T224A/A468T/C560T/G561T/A611G/T612C/T772A/A774T/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1216T/T1218G/C1276T/T1278C AHB2 protein: T187I/Y204C/N226I/L258I/M321I/K324I/N326C/K381R/ 28.70 98 K389I/P456L gene: C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/ G963T/A971T/A972T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB2 protein: T187I/N226I/M321I/K324I/N326C 23.30 99 gene: C560T/G561T/A677T/C678T/G963T/A971T/A972T/A976T/ A977G/T978C AHB3 protein: V75E/T164I/T187I/N226I/L258I/M285L/N326C/K389I/ 36.51 00 I406L gene: T224A/C491T/A492T/C560T/G561T/A677T/C678T/T772A/ A774T/A853C/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G AHB3 protein: V75E/T187I/M285L/M321I/K324I/N326C/R426C 21.11 01 gene: T224A/C560T/G561T/A853C/G963T/A971T/A972T/A976T/ A977G/T978C/C1276T/T1278C AHB3 protein: V75E/T187I/L258I/M285L/N326C/K389I 29.14 02 gene: T224A/C560T/G561T/T772A/A774T/A853C/A976T/A977G/ T978C/A1166T/A1167C AHB3 protein: T164I/T187I/M285L/N326C/K381R/R426C 49.63 03 gene: C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/ A1141C/A1142G/A1143T/C1276T/T1278C AHB3 protein: T164I/T187I/N326C/K381R/I406L 38.86 04 gene: C491T/A492T/C560T/G561T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1216T/T1218G AHB3 protein: T164I/T187I/M285L/N326C/K389I 26.50 05 gene: C491T/A492T/C560T/G561T/A853C/A976T/A977G/T978C/ A1166T/A1167C AHB3 protein: D20V/L156F/T164I/T187I/L258I/M321I/N326C/K381R/ 13.11 06 K389I/I406L/R426C gene: A59T/C60G/A468T/C491T/A492T/C560T/G561T/T772A/ A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB3 protein: D20V/L156F/T187I/M321FK324I/N326C/K389I/R426C/P456L 17.58 07 gene: A59T/C60G/A468T/C560T/G561T/G963T/A971T/A972T/ A976T/A977G/T978C/A1166T/A1167C/C1276T/T1278C/ C1366T/C1367T/A1368G AHB3 protein: T187I/N226I/L258I/N326C/K381R/R426C 25.20 08 gene: C560T/G561T/A677T/C678T/T772A/A774T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB3 protein: T187I/N226I/M285L/N326C/K389I/R426C/P456L 44.86 09 gene: C560T/G561T/A677T/C678T/A853C/A976T/A977G/T978C/ A1166T/A1167C/C1276T/T1278C/C1366T/C1367T/A1368G AHB3 protein: D20V/T164I/T187I/M321I/N326C/K381R/R426C 13.36 10 gene: A59T/C60G/C491T/A492T/C560T/G561T/G963T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB3 protein: T187I/M321I/N326C/K381R/K389I/R426C 22.82 11 gene: C560T/G561T/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1276T/T1278C AHB3 protein: T164I/T187I/Y204C/N226I/L258I/M285L/K324I/N326C/ 40.49 12 K389I gene: C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/ T772A/A774T/A853C/A971T/A972T/A976T/A977G/T978C/ A1166T/A1167C AHB3 protein: Q89H/T187I/Y204C/N226I/N326C/K389I/I406L/R426C 12.46 13 gene: A267T/C560T/G561T/A611G/T612C/A677T/C678T/A976T/ A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/ T1278C AHB3 protein: V75E/L156F/T187I/N226I/K324I/N326C/I406L/R426C 41.37 14 gene: T224A/A468T/C560T/G561T/A677T/C678T/A971T/A972T/ A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C AHB3 protein: Q89H/L156F/T187FY204C/M285L/M321I/N326C/K389I/ 37.44 15 I406L gene: A267T/A468T/C560T/G561T/A611G/T612C/A853C/G963T/ A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G AHB3 protein: Q89H/T187I/Y204C/N326C/P456L 42.78 16 gene: A267T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/ C1366T/C1367T/A1368G AHB3 protein: D20V/Q89H/L156F/T187I/L258I/M321I/N326C/K381R/P456L 21.08 17 gene: A59T/C60G/A267T/A468T/C560T/G561T/T772A/A774T/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ C1366T/C1367T/A1368G AHB3 protein: L156F/T187I/Y204C/M321I/N326C/K389I/R426C 31.34 18 gene: A468T/C560T/G561T/A611G/T612C/G963T/A976T/A977G/ T978C/A1166T/A1167C/C1276T/T1278C AHB3 protein: D20V/V75E/L156F/T187I/Y204C/M285L/N326C/K381R/ 27.27 19 K389I gene: A59T/C60G/T224A/A468T/C560T/G561T/A611G/T612C/ A853C/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C AHB3 protein: D20V/TI87I/L258I/1406L/N326C/R426C 24.71 20 gene: A59T/C60G/C560T/G561T/T772A/A774T/A1216T/T1218G/ A976T/A977G/T978C/C1276T/T1278C AHB3 protein: D20V/V75E/L156F/T187I/M321I/K324I/N326C/R426C/ 11.15 21 P456L gene: A59T/C60G/T224A/A468T/C560T/G561T/G963T/A971T/ A972T/A976T/A977G/T978C/C1276T/T1278C/C1366T/ C1367T/A1368G AHB3 protein: T164I/T187I/N226I/L258I/M285L/M321I/K324I/N326C/ 19.45 22 K381R/K389I/P456L gene: C491T/A492T/C560T/G561T/A677T/C678T/T772A/A774T/ A853C/G963T/A971T/A972T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB3 protein: V75E/L156F/T187I/M285L/M321I/N326C/I406L/R426C 23 gene: T224A/A468T/C560T/G561T/A853C/G963T/A976T/A977G/ 15.24 T978C/A1216T/T1218G/C1276T/T1278C AHB3 protein: T164I/T187I/Y2O4C/N226I/L258I/M285L/M321I/K324I/ 34.32 24 N326C/I406L gene: C491T/A492T/C560T/G561T/A611G/T612C/A677T/C678T/ T772A/A774T/A853C/G963T/A971T/A972T/A976T/A977G/ T978C/A1216T/T1218G AHB3 protein: T187I/N326C/K381R/K389I/I406L/R426C 27.33 25 gene: C560T/G561T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/A1216T/T1218G/C1276T/T1278C AHB3 protein: T164I/T187I/N226I/N326C/R426C 27.55 26 gene: C491T/A492T/C560T/G561T/A677T/C678T/A976T/A977G/ T978C/C1276T/T1278C AHB3 protein: V75E/T164I/T187I/N226I/M321I/N326C/K389I 37.66 27 gene: T224A/C491T/A492T/C560T/G561T/A677T/C678T/G963T/ A976T/A977G/T978C/A1166T/A1167C AHB3 protein: T187I/Y204C/N326C/R426C/P456L 49.60 28 gene: C560T/G561T/A611G/T612C/A976T/A977G/T978C/C1276T/ T1278C/C1366T/C1367T/A1368G AHB3 protein: V75E/Q89H/T187I/Y204C/K324I/N326C/I406L 25.48 29 gene: T224A/A267T/C560T/G561T/A611G/T612C/A971T/A972T/ A976T/A977G/T978C/A1216T/T1218G AHB3 protein: D20V/T187I/N226I/L258I/N326C/K389I/I406L 19.42 30 gene: A59T/C60G/C560T/G561T/A677T/C678T/T772A/A774T/ A976T/A977G/T978C/A1166T/A1167C/A1216T/T1218G AHB3 protein: D20V/T164I/T187I/M285L/M321I/K324I/N326C/I406L 35.80 31 gene: A59T/C60G/C491T/A492T/C560T/G561T/A853C/G963T/ A971T/A972T/A976T/A977G/T978C/A1216T/T1218G AHB3 protein: D20V/T187I/L258I/M321I/N326C/K381R/K389I/R426C 37.14 32 gene: A59T/C60G/C560T/G561T/T772A/A774T/G963T/A976T/ A977G/T978C/A1141C/A1142G/A1143T/A1166T/A1167C/ C1276T/T1278C AHB3 protein: V75E/L156F/T187I/L258I/K324I/N326C 22.86 33 gene: T224A/A468T/C560T/G561T/T772A/A774T/A971T/A972T/ A976T/A977G/T978C AHB3 protein: V75E/T164I/T187I/Y204C/L258I/M321I/N326C/K381R/ 31.73 34 K389I/I406L gene: T224A/C491T/A492T/C560T/G561T/A611G/T612C/T772A/ A774T/G963T/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/A1216T/T1218G AHB3 protein: D20V/V75E/Q89H/T164I/T187I/N226I/M285L/M321I/ 31.88 35 N326C/K389I/I406L gene: A59T/C60G/T224A/A267T/C491T/A492T/C560T/G561T/ A677T/C678T/A853C/G963T/A976T/A977G/T978C/A1166T/ A1167C/A1216T/T1218G AHB3 protein: V75E/Q89H/L156F/T164I/T187I/N226I/M321I/N326C/I406L 14.39 36 gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A677T/ C678T/G963T/A976T/A977G/T978C/A1216T/T1218G AHB3 protein: D20V/T187I/N226I/L258I/N326C/I406L/R426C 21.47 37 gene: A59T/C60G/C560T/G561T/A677T/C678T/T772A/A774T/ A976T/A977G/T978C/A1216T/T1218G/C1276T/T1278C AHB3 protein: L156F/T187FY204C/K389I/N326C/R426C 43.81 38 gene: A468T/C560T/G561T/A611G/T612C/A1166T/A1167C/A976T/ A977G/T978C/C1276T/T1278C AHB3 protein: T187I/Y204C/L258I/M321I/N326C/R426C 12.62 39 gene: C560T/G561T/A611G/T612C/T772A/A774T/G963T/A976T/ A977G/T978C/C1276T/T1278C AHB3 protein: D20V/T187I/N326C/K389I/R426C 23.80 40 gene: A59T/C60G/C560T/G561T/A976T/A977G/T978C/A1166T/ A1167C/C1276T/T1278C AHB3 protein: T187I/L258I/M285L/N326C/R426C 40.73 41 gene: C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/ C1276T/T1278C AHB3 protein: D20V/T164I/T187I/M285L/M321I/N326C/K381R/K389I/ 27.54 42 I406L gene: A59T/C60G/C491T/A492T/C560T/G561T/A853C/G963T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1166T/ A1167C/A1216T/T1218G AHB3 protein: Q89H/L156F/T164I/T187I/Y204C/M285L/M321I/N326C/ 39.32 43 K389I/I406L gene: A267T/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A853C/G963T/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G AHB3 protein: T187I/L258I/M285L/M321I/N326C/K381R 12.88 44 gene: C560T/G561T/T772A/A774T/A853C/G963T/A976T/A977G/ T978C/A1141C/A1142G/A1143T AHB3 protein: T187I/L258I/N326C/K381R/I406L/R426C 21.82 45 gene: C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1216T/T1218G/C1276T/T1278C AHB3 protein: D20V/T164I/T187I/M321I/N326C 15.96 46 gene: A59T/C60G/C491T/A492T/C560T/G561T/G963T/A976T/ A977G/T978C AHB3 protein: Q89H/L156F/T164I/T187I/M285L/N326C/K389I/I406L/ 40.82 47 R426C gene: A267T/A468T/C491T/A492T/C560T/G561T/A853C/A976T/ A977G/T978C/A1166T/A1167C/A1216T/T1218G/C1276T/ T1278C AHB3 protein: V75E/L156F/T164I/T187I/M321I/N326C 33.50 48 gene: T224A/A468T/C491T/A492T/C560T/G561T/G963T/A976T/ A977G/T978C AHB3 protein: D20V/T187I/Y204C/N226I/N326C/K381R/I406L/R426C 29.27 49 gene: A59T/C60G/C560T/G561T/A611G/T612C/A677T/C678T/ A976T/A977G/T978C/A1141C/A1142G/A1143T/A1216T/ T1218G/C1276T/T1278C AHB3 protein: D20V/Q89H/L156F/T187I/Y204C/N226I/M285L/K324I/ 40.05 50 N326C/K381R/K389I gene: A59T/C60G/A267T/A468T/C560T/G561T/A611G/T612C/ A677T/C678T/A853C/A971T/A972T/A976T/A977G/T978C/ A1141C/A1142G/A1143T/A1166T/A1167C AHB3 protein: D20V/V75E/Q89H/L156F/T187VY204C/M285L/M321I/ 19.36 51 N326C/K389I gene: A59T/C60G/T224A/A267T/A468T/C560T/G561T/A611G/ T612C/A853C/G963T/A976T/A977G/T978C/A1166T/ A1167C AHB3 protein: D20V/T164I/T187I/N226I/L258I/N326C/K381R/K389I 28.16 52 gene: A59T/C60G/C491T/A492T/C560T/G561T/A677T/C678T/ T772A/A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C AHB3 protein: L156F/T164I/T187I/Y204C/N226I/L258I/M321I/K324I/N326C 27.65 53 gene: A468T/C491T/A492T/C560T/G561T/A611G/T612C/A677T/ C678T/T772A/A774T/G963T/A971T/A972T/A976T/A977G/ T978C AHB3 protein: V75E/Q89H/T164I/T187I/N326C/K381R/K389I 30.65 54 gene: T224A/A267T/C491T/A492T/C560T/G561T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/A1166T/A1167C AHB3 protein: L156F/T187I/N226I/L258I/M285L/M321I/N326C/K381R/ 41.74 55 K389I/I406L gene: A468T/C560T/G561T/A677T/C678T/T772A/A774T/A853C/ G963T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/A1216T/T1218G AHB3 protein: Q89H/T164I/T187I/Y204C/L258I/N326C/K381R 36.40 56 gene: A267T/C491T/A492T/C560T/G561T/A611G/T612C/T772A/ A774T/A976T/A977G/T978C/A1141C/A1142G/A1143T AHB3 protein: D20V/Q89H/T187I/L258I/N326C 41.30 57 gene: A59T/C60G/A267T/C560T/G561T/T772A/A774T/A976T/ A977G/T978C AHB3 protein: V75E/L156F/T187I/M285L/N326C/K389I 53.85 58 gene: T224A/A468T/C560T/G561T/A853C/A976T/A977G/T978C/ A1166T/A1167C AHB3 protein: Q89H/L156F/T187I/Y204C/N226I/L258I/N326C/K389I/ 31.44 59 I406L/R426C gene: A267T/A468T/C560T/G561T/A611G/T612C/A677T/C678T/ T772A/A774T/A976T/A977G/T978C/A1166T/A1167C/ A1216T/T1218G/C1276T/T1278C AHB3 protein: D20V/V75E/L156F/T164I/T187I/Y204C/M285L/N326C/ 40.31 60 K381R/K389I gene: A59T/C60G/T224A/A468T/C491T/A492T/C560T/G561T/ A611G/T612C/A853C/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C AHB3 protein: V75E/Q89H/L156F/T164I/T187I/N226I/N326C/I406L 24.68 61 gene: T224A/A267T/A468T/C491T/A492T/C560T/G561T/A677T/ C678T/A976T/A977G/T978C/A1216T/T1218G AHB3 protein: T187I/M285L/N326C/K381R/K389I/P456L 44.33 62 gene: C560T/G561T/A853C/A976T/A977G/T978C/A1141C/A1142G/ A1143T/A1166T/A1167C/C1366T/C1367T/A1368G AHB3 protein: V75E/Q89H/T164I/T187I/M321I/K324I/N326C/K381R/I406L 44.79 63 gene: T224A/A267T/C491T/A492T/C560T/G561T/G963T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1216T/T1218G AHB3 protein: Q89H/L156F/T187I/L258I/M321I/K324I/N326C 15.05 64 gene: A267T/A468T/C560T/G561T/T772A/A774T/G963T/A971T/ A972T/A976T/A977G/T978C AHB3 protein: T187I/L258I/M285L/N326C/K389I 41.59 65 gene: C560T/G561T/T772A/A774T/A853C/A976T/A977G/T978C/ A1166T/A1167C AHB3 protein: T187I/Y204C/N226I/L258I/N326C/R426C 36.85 66 gene: C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/ A976T/A977G/T978C/C1276T/T1278C AHB3 protein: V75E/Q89H/T164I/T187I/Y204C/M285L/N326C/R426C 53.56 67 gene: T224A/A267T/C491T/A492T/C560T/G561T/A611G/T612C/ A853C/A976T/A977G/T978C/C1276T/T1278C AHB3 protein: T187I/Y204C/N226I/L258I/N326C 13.47 68 gene: C560T/G561T/A611G/T612C/A677T/C678T/T772A/A774T/ A976T/A977G/T978C AHB3 protein: D20V/L156F/T164I/T187I/N226I/L258I/M285L/N326C/ 32.02 69 I406L/R426C gene: A59T/C60G/A468T/C491T/A492T/C560T/G561T/A677T/ C678T/T772A/A774T/A853C/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C AHB3 protein: T187I/Y204C/L258I/N326C/K381R 18.37 70 gene: C560T/G561T/A611G/T612C/T772A/A774T/A976T/A977G/ T978C/A1141C/A1142G/A1143T AHB3 protein: D20V/T187I/N326C/K381R/I406L 22.23 71 gene: A59T/C60G/C560T/G561T/A976T/A977G/T978C/A1141C/ A1142G/A1143T/A1216T/T1218G AHB3 protein: D20V/V75E/T187I/N226I/K324I/N326C/K381R/K389I/ 34.19 72 P456L gene: A59T/C60G/T224A/C560T/G561T/A677T/C678T/A971T/ A972T/A976T/A977G/T978C/A1141C/A1142G/A1143T/ A1166T/A1167C/C1366T/C1367T/A1368G AHB3 protein: D20V/Q89H/T187I/L258I/M321I/K324I/N326C/K389I 35.87 73 gene: A59T/C60G/A267T/C5 60T/G5 61T/T772A/A774T/G963T/ A971T/A972T/A976T/A977G/T97 8C/A1166T/A1167C AHB3 protein: T164I/T187I/Y204C/M285L/N326C/R426C 50.12 74 gene: C491T/A492T/C560T/G561T/A611G/T612C/A853C/A976T/ A977G/T978C/C1276T/T1278C AHB3 protein: L156F/T187I/Y204C/N326C/K381R 40.72 75 gene: A468T/C560T/G561T/A611G/T612C/A976T/A977G/T978C/ A1141C/A1142G/A1143T AHB3 protein: T187I/L258I/N326C/K389I/I406L/R426C 32.68 76 gene: C560T/G561T/T772A/A774T/A976T/A977G/T978C/A1166T/ A1167C/A1216T/T1218G/C1276T/T1278C AHB3 protein: L156F/T187I/M285L/N326C/K389I 45.87 77 gene: A468T/C560T/G561T/A853C/A976T/A977G/T978C/A1166T/ A1167C AHB3 protein: D20V/T164I/T187I/L258I/K324I/N326C 20.73 78 gene: A59T/C60G/C491T/A492T/C560T/G561T/T772A/A774T/ A971T/A972T/A976T/A977G/T978C AHB3 protein: V75E/L156F/T164I/T187I/Y204C/N226I/L258I/M285L/ 48.01 79 M321I/K324I/N326C gene: T224A/A468T/C491T/A492T/C560T/G561T/A611G/T612C/ A677T/C678T/T772A/A774T/A853C/G963T/A971T/A972T/ A976T/A977G/T978C AHB3 protein: V75E/T187I/M285L/K324I/N326C/K381R/R426C 31.16 80 gene: T224A/C560T/G561T/A853C/A971T/A972T/A976T/A977G/ T978C/A1141C/A1142G/A1143T/C1276T/T1278C AHB3 protein: V75E/T187I/N226I/M285L/N326C 45.50 81 gene: T224A/C560T/G561T/A677T/C678T/A853C/A976T/A977G/ T978C AHB3 protein: Q89H/L156F/T187I/N326C/K381R 39.12 82 gene: A267T/A468T/C560T/G561T/A976T/A977G/T978C/A1141C/ A1142G/A1143T AHB3 protein: D20V/T187I/K324I/N326C/I406L 28.08 83 gene: A59T/C60G/C560T/G561T/A971T/A972T/A976T/A977G/ T978C/A1216T/T1218G AHB3 protein: D20V/T187I/M321I/N326C/K389I 33.93 84 gene: A59T/C60G/C560T/G561T/G963T/A976T/A977G/T978C/ A1166T/A1167C AHB3 protein: V75E/L156F/T187I/N326C/R426C 28.65 85 gene: T224A/A468T/C560T/G561T/A976T/A977G/T978C/C1276T/ T1278C AHB3 protein: T187I/Y204C/N326C/I406L/R426C 16.04 86 gene: C560T/G561T/A611G/T612C/A976T/A977G/T978C/A1216T/ T1218G/C1276T/T1278C

(31) Note: numbering of protein substitutions starts from the N-terminus of the amino acid sequence shown in sequence 2; and numbering of gene substitutions starts from the 5′ end of the nucleotide sequence shown in sequence 1.

(32) The results show that the enzyme activity of the obtained mutants increased by 5-45 times compared with the wild type aspartase.

(33) VIII. Determination of pH Spectrum

(34) By adjusting the amount of ammonia in the substrate, 400 mM substrates with different pH values (7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, and 10.5) were prepared.

(35) After mixing 25 mg of crude enzyme proteins of wild type aspartase or variants thereof with different pH substrates, stirring at 40° C. for 1.0 h of the reaction, and the activity of catalyzed acrylic acid ammoniation was determined. To determine the relative activity, the activity measured at pH 8.0 was set to 100%, and the results are shown in Table 2.

(36) TABLE-US-00003 TABLE 2 pH spectrum measurement results of aspartase variants pH values Variants 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 WT 0.21 0.46 1.00 0.81 0.59 0.32 0.27 0.19 AHB001 0.39 0.71 1.00 0.91 0.82 0.69 0.54 0.39 AHB032 0.59 0.83 1.00 1.02 0.92 0.72 0.62 0.33 AHB063 0.61 0.81 1.00 1.17 0.87 0.86 0.71 0.46 AHB085 0.50 0.80 1.00 1.08 0.80 0.76 0.69 0.51 AHB116 0.44 0.84 1.00 1.02 0.92 0.81 0.66 0.29 AHB148 0.46 0.74 1.00 1.15 0.95 079 0.73 0.54 AHB229 0.45 0.84 1.00 0.99 0.82 0.76 0.62 0.44 AHB244 0.51 0.86 1.00 1.02 0.79 0.69 0.61 0.36 AHB294 0.52 0.78 1.00 1.01 0.89 0.78 0.55 0.41 AHB327 0.43 0.73 1.00 0.95 0.85 0.81 0.73 0.49 AHB357 0.50 0.69 1.00 1.26 1.14 0.90 0.81 0.63 AHB373 0.62 0.77 1.00 1.00 0.91 0.73 0.55 0.42

(37) The results showed that compared with the wild type aspartase, the pH spectrum of the obtained variants was significantly broadened, and the optimal pH value of some variants for catalyzing substrate reactions increased to about 8.5-9.0.

(38) IX. Determination of Temperature Spectrum

(39) Mixing 25 mg of crude protein of wild type aspartase or variants thereof with 1.0 mL of pH 8.0, 400 mM substrate, and stirring at different temperatures (30° C., 35° C., 40° C., 45° C., 50° C., 55° C.), respectively for reacting 1.0 h. Then, the activity of catalyzing the ammoniation of acrylic acid is measured. To determine the relative activity, the activity measured at 40° C. was set to 100%, and the results are shown in Table 3.

(40) TABLE-US-00004 TABLE 3 Temperature spectrum measurement results of aspartase variants temperature 30° 35° 37° 40° 45° 50° 55° Variants C. C. C. C. C. C. C. WT 0.41 0.62 0.55 1.00 0.79 0.62 0.20 AHB001 0.39 0.67 0.79 1.00 0.88 0.81 0.76 AHB032 0.56 0.84 0.89 1.00 1.06 0.86 0.84 AHB063 0.20 0.63 0.96 1.00 1.05 0.82 0.75 AHB085 0.52 0.72 0.84 1.00 0.89 0.89 0.76 AHB116 0.76 0.87 1.10 1.00 1.13 0.92 0.81 AHB148 0.70 0.90 0.91 1.00 1.34 0.96 0.79 AHB229 0.58 0.86 0.99 1.00 0.99 0.91 0.87 AHB244 0.78 1.05 0.89 1.00 1.04 1.06 0.84 AHB294 0.51 0.80 0.95 1.00 1.00 0.92 0.84 AHB327 0.67 0.88 1.01 1.00 1.26 1.09 0.79 AHB357 0.31 0.54 0.73 1.00 1.31 1.14 0.98 AHB373 0.77 1.03 0.96 1.00 0.99 1.03 0.87

(41) The results showed that compared with the wild type aspartase, the temperature spectrum of the obtained variants was significantly broadened, and the optimal temperature for catalyzing substrate of some variants increased to 45° C.-50° C.

(42) X. Determination of Thermal Stability

(43) After incubating 25 mg of crude enzyme protein of wild type aspartase or variants thereof at 40° C. for 4 hours, mixing with 1.0 mL of pH 8.0 and 400 mM substrate, and stirring at 40° C. for 1.0 h of reaction. Then, the activity of catalyzing the ammoniation of acrylic acid is measured. The experimental group is one or more (several) variants of aspartase, and the control group is wild type aspartase. The results are shown in Table 4.

(44) TABLE-US-00005 TABLE 4 Measurement results of thermal stability of aspartase variants Enzyme activity Variants retention WT 21.40% AHB001 39.03% AHB032 60.64% AHB063 56.16% AHB085 50.88% AHB116 58.08% AHB148 44.40% AHB229 52.24% AHB244 57.52% AHB294 51.36% AHB327 54.80% AHB357 64.08% AHB373 60.64%

(45) The results showed that the thermal stability of the obtained variant increased significantly compared with the wild-type aspartase. After incubating at 40° C. for 4 hours, the enzyme activity retention rate increased by about 2-3 times.

(46) XI. Determination of Acrylic Acid Conversion

(47) Mixing 25 mg of crude enzyme proteins of wild type aspartase or variants thereof was with 1.0 mL of substrate (with a concentration of 400 mM) at pH 8.5, and stirring at 45° C. for 0-5 h of reaction. Acrylic acid residues are detected by liquid chromatogram, to calculate the conversion ratio. The results are shown in Table 5.

(48) TABLE-US-00006 TABLE 5 Determination of acrylic acid conversion ratio Var- conversion ratio after different reaction time iants 0 h 0.5 h 1.0 h 2.0 h 3.0 h 4.0 h 5.0 h WT 0%  0.54%  0.86%  0.90%  1.03%  1.26%  1.34% AHB- 0% 10.62% 30.64% 45.39% 53.64% 62.32% 68.33% 001 AHB- 0% 19.44% 51.15% 69.54% 84.21% 88.43% 91.12% 063 AHB- 0% 31.26% 49.75% 65.41% 77.34% 82.22% 84.73% 116 AHB- 0% 26.41% 59.72% 74.49% 87.32% 92.25% 93.67% 357 AHB- 0% 23.63% 54.26% 72.91% 83.69% 89.24% 92.13% 373

(49) As shown in Table 5, after 5 hours of reaction, the conversion ratio of the wild type aspartase substrate was only 1.34%, the substrate conversion ratio of the AHB001 variant reached 68.33%, and the substrate conversion ratios of the other shown variants reached greater than 84%.

(50) The invention described and claimed herein is not limited to the specific embodiments disclosed herein, as the above embodiments are intended to be illustrative of a particular aspect of the invention and any equivalent embodiments are intended to be included in the scope of the invention.

INDUSTRIAL APPLICATION

(51) Compared with the wild type parent aspartase, the aspartase variant provided in the present invention has improved catalytic activity of the ammoniation of acrylic acid, and has better thermal stability and pH spectrum, and the conversion ratio of acrylic acid can be improved significantly in the reaction of the ammoniation of acrylic acid. The aspartase variant provided in the present invention has important application value for catalyzing the ammoniation of acrylic acid to generate β-alanine, and then meeting the needs of industrialization.